Variants of C-type natriuretic peptide

ABSTRACT

The present disclosure provides variants of C-type natriuretic peptide (CNP), pharmaceutical compositions comprising CNP variants, and methods of making CNP variants. The CNP variants are useful as therapeutic agents for the treatment of diseases responsive to CNP, including but not limited to bone-related disorders, such as skeletal dysplasias (e.g., achondroplasia), and vascular smooth muscle disorders (e.g., restenosis and arteriosclerosis).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the priority and benefit of U.S.Provisional Application No. 61/254,563, filed on Oct. 23, 2009, and U.S.Provisional Application No. 61/180,112, filed on May 20, 2009, thedisclosure of each of which is incorporated herein by reference in itsentirety.

FIELD OF THE DISCLOSURE

The field of the disclosure relates, in general, to variants of C-typenatriuretic peptide (CNP), compositions comprising CNP variants, methodsof making CNP variants, and methods of using CNP variants to treatdisorders responsive to CNP, including but not limited to bone-relateddisorders such as skeletal dysplasias (e.g., achondroplasia) andvascular smooth muscle disorders.

BACKGROUND OF THE DISCLOSURE

The natriuretic peptide family consists of three structurally relatedpeptides: atrial natriuretic peptide (ANP) (Genbank Accession No.NP_(—)006163, for the ANP precursor protein, NPPA), brain natriureticpeptide (BNP) (GenBank Accession No. NP_(—)002512, for the BNP precursorprotein, NPPB), and C-type natriuretic peptide (CNP) (Biochem. Biophys.Res. Commun., 168: 863-870 (1990) (GenBank Accession No. NP_(—)077720,for the CNP precursor protein, NPPC) (J. Hypertens., 10: 907-912(1992)). These small, single chain peptides (ANP, BNP, CNP) have a17-amino acid loop structure (Levin et al., N. Engl. J. Med., 339:863-870 (1998)) and have important roles in multiple biologicalprocesses. ANP and BNP bind to and activate the natriuretic peptidereceptor A (NPR-A), also termed guanalyl cyclase A (GC-A), resulting inhigher intracellular cyclic guanosine monophosphate (cGMP) levels.Likewise, CNP interacts with NPR-B (GC-B) to stimulate the generation ofcGMP (J. Hypertens., 10: 1111-1114 (1992)). A third type of receptor,NPR-C, binds each of the natriuretic peptides with high affinity andfunctions primarily to capture the peptides from the extracellularcompartment and deposit the peptides into lysosomes, where they aredegraded (Science, 238: 675-678 (1987)). ANP and BNP are producedprimarily within the muscle cells of the heart, and are believed to haveimportant roles in cardiovascular homeostasis (Science, 252: 120-123(1991)). CNP is expressed more widely, including in the central nervoussystem, reproductive tract, bone and endothelium of blood vessels(Hypertension, 49: 419-426 (2007)).

In humans, CNP is initially produced from the natriuretic peptideprecursor C(NPPC) gene as a single chain 126-amino acid pre-propolypeptide (Biochem. Biophys. Res. Commun., 168: 863-870 (1990)).Removal of the signal peptide yields pro-CNP, and further cleavage bythe endoprotease furin generates an active 53-amino acid peptide(CNP-53), which is secreted and cleaved again by an unknown enzyme toproduce the mature 22-amino acid peptide (CNP-22) (Wu, J. Biol. Chem.278: 25847-852 (2003)). CNP-53 and CNP-22 differ in their distribution,with CNP-53 predominating in tissues, while CNP-22 is mainly found inplasma and cerebrospinal fluid (J. Alfonzo, Recept. Signal. Transduct.Res., 26: 269-297 (2006)). The predominant CNP form in cartilage isunknown. Both CNP-53 and CNP-22 bind similarly to NPR-B. Furthermore,they both induce cGMP production in a dose-dependent and similar fashion(V T Yeung, Peptides, 17: 101-106 (1996)).

Natural CNP gene and polypeptide have been previously described. U.S.Pat. No. 5,352,770 discloses isolated and purified CNP-22 from porcinebrain identical in sequence to human CNP and its use in treatingcardiovascular indications. U.S. Pat. No. 6,034,231 discloses the humangene and polypeptide of proCNP (126 amino acids) and the human CNP-53gene and polypeptide.

Clearance of CNP from the extracellular space occurs through the actionof membrane-bound neutral endopeptidase (NEP), which rapidly degradesCNP (Biochem. J., 291 (Pt 1): 83-88 (1993)), and through NPR-C, whichbinds to and deposits CNP into lysosomes, where CNP is degraded. CNP hasbeen shown to have an in vivo half-life of 2.6 min in the normal human(J. Clin. Endocrinol. Metab., 78: 1428-35 (1994)). The low plasmaconcentration of CNP (J. Bone Moner. Res., 19 (Suppl. 1) S20 (2004)) andits co-expression with NPR-B in a number of tissues suggests that CNPfunctions primarily through an autocrine/paracrine mechanism.

As stated above, CNP binds to and activates natriuretic peptide receptorB (NPR-B), also termed guanylyl cyclase B (GC-B), resulting in higherintracellular cyclic guanosine monophosphate (cGMP) levels. Downstreamsignaling mediated by cGMP generation influences a diverse array ofbiological processes that include endochondral ossification.Accordingly, elevated or depressed levels of any of the components inthis pathway may lead to aberrant bone growth. For example, knockout ofeither CNP or NPR-B in mouse models results in animals having a dwarfedphenotype with shorter long bones and vertebrae. Mutations in humanNPR-B that block proper CNP signaling have been identified and result indwarfism (Olney, et al., J. Clin. Endocrinol. Metab. 91(4): 1229-1232(2006); Bartels, et al., Am. J. Hum. Genet. 75: 27-34 (2004)). Incontrast, mice engineered to produce elevated levels of CNP displayelongated long bones and vertebrae.

Achondroplasia is a result of an autosomal dominant mutation in the genefor fibroblast growth factor receptor 3 (FGFR-3), which causes anabnormality of cartilage formation. FGFR-3 normally has a negativeregulatory effect on chondrocyte growth, and hence bone growth. Inachondroplasia, the mutated form of FGFR-3 is constitutively active,which leads to severely shortened bones. Both chondrocyte proliferationand differentiation appear to be disturbed, leading to remarkably shortgrowth plate cartilage (P. Krejci et al., J. Cell Sci. 118: 5089-5100(2005)). Endochondral ossification is the process that governslongitudinal long-bone growth. There are four zones of the growthplate—resting, proliferative, hypertrophic and zone of calcification. Inthe growth plate, NPR-B is expressed by proliferative cells while NPR-Cis expressed by hypertrophic cells (Yamashite et al., J. Biochem. 127:177-179 (2000)). In normal endochondral bone growth, chondrocytesorganize in columns and proliferate in the proliferative zone of thegrowth plate. These columns are disorganized in achondroplasia patients.Additionally, the hypertrophic zone is where the cells become large andeventually apoptose (lyse), leading to osteocyte invasion andmineralization. The hypertrophic chondrocytes and the overall size ofthe zone are much smaller in achondroplasia patients than in normalpatients. CNP is an agonist for NPR-B, a positive regulator ofchondrocyte and bone growth. Downstream signaling of CNP/NPR-B inhibitsthe FGFR-3 pathway at the level of mitogen-activated protein kinase (MAPK). Inhibition at MAP K promotes proliferation and differentiation ofthe chondrocytes in the proliferative and hypertrophic zones of thegrowth plate, resulting in bone growth.

In humans activating mutations of FGFR-3 are the primary cause ofgenetic dwarfism. Mice having activated FGFR-3 serve as a model ofachondroplasia, the most common form of the skeletal dysplasias, andoverexpression of CNP rescues these animals from dwarfism. Accordingly,CNP and functional variants of CNP are potential therapeutics fortreatment of the various skeletal dysplasias.

Therapeutic use of CNP is currently limited by its short plasmahalf-life, which has been shown to be 2.6 minutes in vivo in humans (J.Clin. Endocrinol. Metab., 78: 1428-35 (1994)). To increase CNPconcentration above intrinsic levels (about 5 pM) typically found inhuman plasma, continuous infusion has been necessary in all human andanimal studies using systemically administered CNP. A CNP variant havinga longer in vivo serum half-life and exhibiting similar or improvedactivity to that of wild-type CNP is important for a sustainabletherapeutic strategy. Two mechanisms by which the half-life of CNP isreduced in human plasma are degradation by neutral endopeptidase (NEP)and clearance by natriuretic peptide receptor C(NPR-C) (Growth Horm. &IGF Res., 16: S6-S14 (2006)). Modifications of peptides reportedly canimprove resistance to endopeptidase and exopeptidase cleavage (AminoAcids, 30: 351-367 (2006); Curr. Opin. Biotech., 17: 638-642 (2006)).

The biological activities of various analogs and derivatives of CNP havebeen evaluated. By substituting S-methyl Cys in place of both Cys₆ andCys₂₂, cyclization of the peptide via a Cys6-Cys22 disulfide linkage wasreportedly shown to be important for the activity of CNP in stimulatingcGMP formation (Biochem. Biophys. Res. Comm., 183: 964-969 (1992), alsousing alanine scanning to identify amino acids important for CNPfunctionality). A significant additional enhancement of activityreportedly results from the combined presence of the amino acids Leu₉,Lys₁₀, and Leu₁₁. U.S. Pat. No. 5,434,133 describes CNP analogscomprising CNP-22 with substitutions at amino acid position 6, 7, 9, 11,or 22, wherein the amino acid is selected from Cys or Pmp(pentacyclomercaptopropionic acid) at position 6, Phe, 4-chloro-Phe,4-fluoro-Phe, 4-nitro-Phe, or Cha (3-cyclohexyl-Ala) at position 7, Gly,Val, Aib, or tLeu at position 9, Leu or Ile at position 11, and Cys orPmp at position 22.

U.S. Patent Publication No. 2004/0138134 (now U.S. Pat. No. 7,276,481)describes CNP variants comprising amino acids Cys₆ to Cys₂₂ of CNP-22(“CNP-17”) which include at least one substitution for another naturalamino acid at position 9, 10, 11, 16, 17, 19, or 20, CNP variants withinsertions and deletions, such as addition of a His residue at thereported primary site of NEP cleavage, between Cys₆ and Phe₇, andmethods of using such variants for increasing the size of a bone growthplate in abnormal bone and elongation of an abnormal bone. However, nosignificant gains in activity as measured by cGMP production wereobtained for these variants, and activity was diminished for nearly allof the variants, as observed in an in vitro cell-based method (Example7). Further no supportive data, such as for example in vitro stabilityor in vivo determination of improved pharmacokinetics (PK) were providedto substantiate the asserted NEP resistance and NPR-C resistance of theCNP analogs. U.S. Pat. No. 6,743,425 discloses substances for treatingachondroplasia which activate NPR-B/GC-B and are peptides or lowmolecular weight compounds, including the C-type natriuretic peptidesCNP-22 and CNP-53. PCT Publication No. WO 94/20534 discloses a chimeraof CNP-22 and the 5-amino acid C-terminus of ANP designated as thevasonatrin peptide (VNP), a limited number of amino acid substitutionsand cyclic chimeric peptides that result from formation of a disulfideor double bond.

Approaches for improving the half-life of other natriuretic peptidefamily members include decreasing the affinity of ANP for NPR-C (U.S.Pat. No. 5,846,932), utilizing pentapeptide antagonists of NPR-C (WO00/61631), and co-administering NEP inhibitors such as thiorphan andcandoxatril (Clin. Exp. Pharma. Physiol., 25: 986-991 (1997), Hyperten.,30: 184-190 (1997)). WO 2004/047871 describes conjugates of BNP and BNPvariants to polyalkylene glycol moieties, sugar moieties, polysorbatemoieties, polycationic moieties, and other hydrophilic polymer moietiesthat reportedly exhibit improved half-life in circulation and reportedlyare useful for the treatment of acute congestive heart failure.

There have been no published reports, however, on a successful strategyfor making CNP resistant to NEP while retaining its functionality.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to variants of C-type natriuretic peptide(CNP) which are useful in the treatment of bone-related disorders (e.g.,achondroplasia) and vascular smooth muscle disorders. The disclosureencompasses CNP variants having increased serum half-life, e.g. as aresult of reduced ability to bind to neutral endopeptidase (NEP),greater resistance to proteolysis by NEP and/or reduced affinity to theclearance natriuretic peptide receptor C(NPR-C), while retaining thefunctionality of CNP.

The wild-type sequence of human CNP-22 (referred to herein as “hCNP22”,“wtCNP22” or “CNP22”) is set forth below:

(SEQ ID NO: 1) (N-terminus) Gly₁-Leu₂-Ser₃-Lys₄-Gly₅-Cys₆-Phe₇-Gly₈-Leu₉-Lys₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂.Positions 6 to 22 of CNP22 form a cyclic domain by means of a disulfidebond between Cys6 and Cys22. The 17-amino acid cyclic structure has beenshown to be important for binding of CNP to NPR-B (Schiller, Biochem.Biophys. Res. Commun., 138: 880-886 (1986)). The amino acid sequence ofpositions 6 to 22 of CNP22 is referred to herein as “CNP17” (SEQ ID NO:2).

CNP is susceptible to NEP cleavage at a number of sites: Cys6-Phe7,Gly8-Leu9, Lys10-Leu11, Arg13-Ile14, Ser16-Met17 and Gly19-Leu20. In oneembodiment, the disclosure encompasses a CNP variant that is (1)modified to increase its overall size or molecular weight, e.g., to arange from about 2.6 kDa or 2.8 kDa to about 4 kDa, 4.2 kDa, 4.4 kDa,4.6 kDa, 4.8 kDa, 5 kDa, 5.2 kDa, 5.4 kDa, 5.6 kDa, 5.8 kDa, 6 kDa, 6.2kDa, 6.4 kDa, or to about 7 kDa, 7.2 kDa or about 8.2 kDa, and/or (2)modified at certain amino acid positions to reduce its susceptibility toNEP cleavage at 1, 2, 3, 4, 5 or all 6 of the sites listed above. Thesize or molecular weight of the CNP variant can be increased by variousmeans, e.g., by conjugating additional amino acids and/or other kinds ofchemical (e.g., natural or synthetic polymeric) groups to the peptidesequence at, e.g., the N-terminus, the C-terminus and/or side chain(s),and/or by using natural amino acids, unnatural amino acids, and/orpeptidomimetics with bulkier side chains. The CNP variant is optionallyfurther conjugated to other functional or structural moieties.Optionally in combination with any of the embodiments described herein,mutation(s) (e.g., substitution(s), addition(s), and/or deletion(s)) maybe introduced to certain position(s) of CNP22 to reduce the CNPvariants' affinity to NPR-C. Further modifications may be made withoutaffecting NEP resistance or CNP activity, e.g., conservativesubstitutions, or other modifications known in the art.

In one embodiment, the CNP variant is represented by the generalformula:(x)-Gly₁-Leu₂-Ser₃-Lys₄-Gly₅-Cys₆-Phe₇-Gly₈-Leu₉-Lys₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂-(z)(SEQ ID NO: 5), wherein:

the CNP variant comprises one or more modified amino acids, which mayresult in modified peptide bonds (e.g., through use of peptide bondisosteres), at a position corresponding to one or more of the followingCNP residues: Gly1, Lys4, Gly5, Cys6, Phe7, Gly8, Leu9, Lys10, Leu11,Ile14, Gly15, Ser16, Met17, Gly19, Leu20 and Gly21; and

(x) and (z) independently may be absent or may be an amino acid sequencederived from a natriuretic polypeptide (e.g., NPPC, ANP, BNP) or anon-natriuretic polypeptide (e.g., human serum albumin (HSA), IgG,etc.).

In an embodiment, the CNP variant includes: (1) a modification at anamino acid position corresponding to one of positions 6, 7 or 8 (Cys6,Phe7 or Gly8) of CNP22, (2) optionally deletion, addition and/orsubstitution of any or all of the amino acids at positions 1-5 (Gly1,Leu2, Ser3, Lys4, and Gly5) and (3) optionally up to 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 further modifications (deletions, additions and/orsubstitutions) at positions corresponding to positions 6-22, of which 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 may be conservative substitutions or othersubstitutions described herein or known in the art.

It is understood that a reference to a particular amino acid position bynumber (e.g. position 7 of CNP22) refers to the corresponding amino acidposition in any CNP variant, even if the number of the position in thatCNP variant has changed due to preceding insertions or deletions. Forexample, a reference to “position 7” or “Phe7” would mean thecorresponding position 2 for a CNP variant in which the first five aminoacids had been deleted. Similarly, a reference to “position 7” wouldmean the corresponding position 8 for a CNP variant in which one aminoacid had been added to the N-terminus.

In any of the embodiments described herein, the CNP variant may becyclized through a covalent bond between positions corresponding to 6and 22 of CNP22. It is contemplated that the covalent bond is formedusing any methods known in the art. In another embodiment, the CNPvariant may be cyclized through a covalent bond formed between an aminoacid at or toward the N-terminus and an amino acid at or toward theC-terminus (referred to as “terminal” amino acids for this purpose) ofthe peptide. In one embodiment, the covalent bond is formed between theside chains of the two terminal amino acids or the amino acids atpositions corresponding to 6 and 22 of CNP22. In another embodiment, thecovalent bond is formed between the side chain of one terminal aminoacid and the terminal group of the other terminal amino acid, or betweenthe terminal groups of each terminal amino acid. For example,head-to-tail, side chain-to-side chain, side chain-to-head, or sidechain-to-tail bonds are possible for the covalent bond formed betweenthe terminal amino acids or between the amino acids at positionscorresponding to 6 and 22 of CNP22.

In one embodiment, the disclosure provides a CNP variant having reducedaffinity to NEP, and/or greater resistance to cleavage by NEP and/orincreased in vivo serum half-life, while retaining functionality of CNP(e.g., stimulation of cGMP production). NEP preferably recognizessubstrates smaller than about 3 kDa, due to the limited size of itsactive site cavity (Oefner, J. Mol. Biol., 296: 341-349 (2000)). In anembodiment, the CNP variants are modified to increase their overallmolecular weight to a range from about 2.6 or 2.8 kDa to about 4, 4.6,5, 5.2, 5.8, 6, 6.4 or 7 kDa, e.g., by adding about 0.6 to about 5 kDaof amino acids, hydrophilic or water-soluble polymers, hydrophobic acids(including fatty acids), and/or carbohydrates. In specific exemplaryembodiments, the CNP variants have a molecular weight between about 2.6kDa and about 7 kDa, or between about 2.8 kDa and 6 kDa, or betweenabout 2.8 kDa and about 5.8 kDa. In certain embodiments, at least about0.6, 0.8, 1, 1.2, 1.4, 1.6 or 1.8 kDa, or up to 2, 2.2, 2.4, 2.6, 2.8,3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8 or 5 kDa are added, toincrease the total molecular weight of the CNP variant, e.g., to a rangefrom about 2.6 or 2.8 kDa up to about 4 kDa, 4.2 kDa, 4.4 kDa, 4.6 kDa,4.8 kDa, 5 kDa, 5.2 kDa, 5.4 kDa, 5.6 kDa, 5.8 kDa, 6 kDa, 6.2 kDa, 7.2kDa, 8.2 kDa or higher. In some embodiments, such CNP variants comprisean amino acid sequence at least about 70%, 75%. 80%, 85%, 90%, or 95%identical or homologous to amino acids 6-22 of CNP22. In otherembodiments, such CNP variants comprise a substitution, insertion ordeletion of 1, 2, 3, 4, 5, 6 or 7 amino acids with another natural orunnatural amino acid or peptidomimetic. While both conservative andnon-conservative substitutions or insertions are envisioned at anyposition, introduction of modifications may commence, e.g., byconservative substitutions in regions that have been identified in theart as involved in CNP activity or NPR-B binding, while non-conservativesubstitutions may be made in those regions that have been previouslyshown to be tolerant of modification.

In another embodiment, the CNP variants comprise a CNP having an intactcyclized portion between Cys6 and Cys22, and N-terminal and/orC-terminal tails that contain about 1-40, 1-20, 5-40, 5-35, 10-35,15-35, 5-31, 10-31, or 15-31 amino acids and are fragments derived froma CNP polypeptide and/or a non-CNP polypeptide. In an embodiment, suchCNP variants have a molecular weight in a range from about 2.8 kDa toabout 4, 4.6, 5, 5.2, 5.8, 6, 6.4 or 7 kDa. Non-limiting examples ofsuch CNP variants include wild-type CNP22 or CNP22 with one or moreamino acid substitutions (e.g., a K4R substitution), having anN-terminal and/or C-terminal extension derived from natriuretic peptideprecursor sequences (e.g., ANP, BNP or CNP) from human or other species,a natriuretic peptide precursor C (NPPC) variant with amino acidsubstitutions, additions and/or deletions (e.g., the CNP variants may betruncations of CNP-53 which result in peptides with a molecular weightbetween about 2.8 kDa and 5.8 kDa), or other non-CNP polypeptides suchas, e.g., serum albumin or IgG protein (e.g., the CNP variants may beCNP chimeras containing fragments of serum albumin or IgG from human orother species).

In one embodiment, CNP variants having a total mass characterized by theranges described generally herein, e.g., from about 2.6 kDa or 2.8 kDato about 6 or 7 kDa, designed for increased resistance to NEPdegradation, are represented by the general formula:

-   (x)-Gly₁-Leu₂-Ser₃-(b)₄-Gly₅-Cys₆-Phe₇-Gly₈-Leu₉-(h)₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂-(z)    (SEQ ID NO: 6), wherein:

(x) is a synthetic or natural polymeric group, or a combination thereof,wherein a non-limiting example of a synthetic polymeric group ispolyethylene glycol (PEG, also called polyethylene oxide (PEO)), and anon-limiting example of a natural polymeric group is an amino acidsequence containing from 1 to 35 amino acids and derived from NPPC orvariants thereof with substitutions and/or deletions, ANP, BNP, or othernon-CNP (poly)peptides such as, e.g., serum albumin, IgG, histidine-richglycoproteins, fibronectin, fibrinogen, zinc finger-containingpolypeptides, osteocrin or fibroblast growth factor 2 (FGF2);

(z) may be absent or may be a synthetic or natural polymeric group, or acombination thereof, wherein a non-limiting example of a syntheticpolymeric group is PEG, and a non-limiting example of a naturalpolymeric group is an amino acid sequence derived from a natriureticpolypeptide (e.g., NPPC, CNP, ANP or BNP) or non-natriuretic polypeptide(e.g., serum albumin or IgG); and

(b) and (h) independently may each be the wild type Lys at that positionor may be replaced with a conservative amino acid substitution or anynatural or unnatural amino acid or peptidomimetic that does not have areactive primary amine on a side chain, including but not limited toArg, Gly, 6-hydroxy-norleucine, citrulline (Cit), Gln, Glu or Ser. Inone embodiment, (b) is Arg. In another embodiment, for improved NEPresistance, (b) is not Gly. In yet another embodiment, (h) is not Arg.

Non-limiting examples of amino acid sequences derived from NPPC orvariants thereof include:

Arg, Glu-Arg, (SEQ ID NO: 7) Gly-Ala-Asn-Lys-Lys, (SEQ ID NO: 8)Gly-Ala-Asn-Arg-Arg, (SEQ ID NO: 9) Gly-Ala-Asn-Pro-Arg, (SEQ ID NO: 10)Gly-Ala-Asn-Gln-Gln, (SEQ ID NO: 11) Gly-Ala-Asn-Ser-Ser, (SEQ ID NO:12) Gly-Ala-Asn-Arg-Gln, (SEQ ID NO: 13) Gly-Ala-Asn-Arg-Met, (SEQ IDNO: 14) Gly-Ala-Asn-Arg-Thr, (SEQ ID NO: 15) Gly-Ala-Asn-Arg-Ser, (SEQID NO: 16) Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn- Ala, (SEQ IDNO: 17) Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn- Ala-Arg, (SEQID NO: 18) Asp-Leu-Arg-Val-Asp-Thr-Lys-Ser-Arg-Ala-Ala-Trp- Ala-Arg,(SEQ ID NO: 19) Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Lys-Lys, (SEQ ID NO: 20)Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala- Asn-Arg-Arg, (SEQ IDNO: 21) Asp-Leu-Arg-Val-Asp-Thr-Lys-Ser-Arg-Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Lys-Lys, and (SEQ ID NO: 22)Asp-Leu-Arg-Val-Asp-Thr-Lys-Ser-Arg-Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Arg-Arg.

Non-limiting examples of amino acid sequences derived from non-CNPpolypeptides such as, e.g., ANP, BNP, serum albumin and IgG include:

(SEQ ID NO: 23) Ser-Leu-Arg-Arg-Ser-Ser; (SEQ ID NO: 24)Asn-Ser-Phe-Arg-Tyr; (SEQ ID NO: 25)Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly; (SEQ ID NO: 26)Met-Val-Gln-Gly-Ser-Gly; (SEQ ID NO: 27) Lys-Val-Leu-Arg-Arg-Tyr; (SEQID NO: 28) Lys-Val-Leu-Arg-Arg-His; (SEQ ID NO: 29)Gly-Gln-His-Lys-Asp-Asp-Asn-Pro-Asn-Leu-Pro-Arg; (SEQ ID NO: 30)Gly-Val-Pro-Gln-Val-Ser-Thr-Ser-Thr; (SEQ ID NO: 31)Gly-Glu-Arg-Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg- Leu-Ser-Gln; and (SEQID NO: 32) Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro- Pro-Ser.

In an embodiment, the N-terminus and/or C-terminus of CNP22 or a variantthereof independently may be conjugated to an amino acid extensioncontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39 or 40 amino acids. In one embodiment, the amino acidextension is derived from NPPC, CNP53, ANP or BNP. In a specificembodiment, the amino acid extension isGln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Lys-Lys (SEQ ID NO:33). In a related embodiment, this 15-amino acid extension is added tothe N-terminus to provide a CNP variant of the formulaGln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Lys-Lys-Gly₁-Leu₂-Ser₃-(b)₄-Gly₅-Cys₆-Phe₇-Gly₈-Leu₉-(h)₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂-(z)(SEQ ID NO: 34).

In one embodiment, the CNP variants comprise wtCNP22 or a variantthereof (e.g., one having addition(s), deletion(s), and/orsubstitution(s) such as, e.g., a K4R substitution) (SEQ ID NO: 35)conjugated at the N-terminus and/or C-terminus to a hydrophilic polymer(e.g., PEG) to increase their overall molecular size to a range fromabout 2.6 kDa or 2.8 kDa to about 4, 5, 6 or 7 kDa. Such CNP variantsare optionally further conjugated at the N-terminus and/or C-terminus toa polymeric group comprising, e.g., amino acids, carbohydrates,hydrophobic acids and/or phospholipids, a non-limiting example of whichis an N-terminal amino acid extension containing 1 to 35, or 5 to 31,amino acids. In an embodiment, a hydrophilic polymeric (e.g., PEG)moiety of at least about 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6 or 1.8 kDa, orup to about 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4,4.6, 4.8 or 5 kDa, is added to the N-terminus and/or C-terminus ofwtCNP22 or a variant thereof.

As shown herein, conjugation of a hydrophilic or water-soluble PEG (orPEO) polymer of about 0.6 kDa or more to CNP22 or variants thereofgenerally increases resistance to NEP cleavage markedly. However,addition of PEG, even as small as 0.6 kDa, to wtCNP22 may reduce CNPfunctionality (e.g., stimulation of cGMP signaling), and addition ofgreater than about 2 or 3 kDa of PEG to CNP22 or variants thereof mayreduce CNP functional activity in a size-dependent manner. But CNPfunctionality (at least comparable to that of wtCNP22) is retained whena PEG (or PEO) polymer of about 0.6 kDa to about 1.2 kDa, or potentiallyto about 2 kDa, is conjugated to a CNP variant having an N-terminalamino acid extension in which at least one relatively large amino acidthat may potentially be positively charged under physiologicalconditions (e.g., arginine) immediately precedes the positioncorresponding to Gly1 of CNP22, such as, e.g., GANRR-CNP22(K4R) (SEQ IDNO: 36), GANPR-CNP22(K4R) (SEQ ID NO: 37), ER-CNP22 (SEQ ID NO: 38),ER-CNP22(K4R) (SEQ ID NO: 39), R-CNP22 (SEQ ID NO: 40) and R-CNP22(K4R)(SEQ ID NO: 41).

Accordingly, in one embodiment, PEGylated CNP variants comprise at theN-terminus of CNP22 or a variant thereof (e.g., one having a K4Rsubstitution) an amino acid extension containing at least 1, 2, 3, 4 or5 amino acids, wherein the PEG polymer is conjugated to the N-terminusof the amino acid-extended CNP variant to result in a total masscharacterized by the ranges described generally herein, e.g., from about2.6 kDa or 2.8 kDa to about 6 or 7 kDa for increased resistance to NEPcleavage. In an embodiment, for enhanced CNP functionality, suchpegylated, amino acid-extended CNP variants contain at least onerelatively large natural or unnatural amino acid that may potentially bepositively charged under physiological conditions, immediately precedingthe position corresponding to Gly1 of CNP22. In a specific embodiment,the pegylated, amino acid-extended CNP variants contain at least onearginine residue immediately preceding the position corresponding toGly1 of CNP22.

In addition to CNP variants conjugated at the N-terminus and/orC-terminus to a hydrophilic or water-soluble polymer such as, e.g., PEG(or PEO), the disclosure encompasses CNP variants conjugated to such apolymer at an internal site. For purposes of brevity here, PEG (or PEO)will be used as a representative example of a hydrophilic orwater-soluble polymer. Various sites of PEGylation of a CNP variant arepossible, including but not limited to: (1) PEGylation only at theN-terminus; (2) PEGylation only at the C-terminus; (3) PEGylation onlyat an internal site (e.g., Lys4); (4) PEGylation at both the N-terminusand the C-terminus; (5) PEGylation at the N-terminus and an internalsite; and (6) PEGylation at the C-terminus and an internal site. Forincreased resistance to NEP degradation and retention of CNPfunctionality, in certain embodiments the total mass of PEGylatedCNPvariants is characterized by the ranges described generally herein,e.g., in the range from about 2.6 kDa or 2.8 kDa to about 4, 5, 6 or 7kDa. In a particular embodiment, CNP17, CNP22, CNP37 (defined below) orvariants thereof (including those having amino acid additions,substitutions and/or deletions) are PEGylated only at the N-terminus. Inanother embodiment, the CNP variants are PEGylated only at an internalsite (e.g., Lys4). In yet another embodiment, the CNP variants arePEGylated at the N-terminus and an internal site (e.g., Lys4). In stillanother embodiment, for better functionality the CNP variants are notPEGylated at a site (e.g., Lys10) within the cyclic domain(corresponding to Cys6 to Cys22 of CNP22). To prevent PEGylation at aninternal site, Lys4 and/or Lys10 can be substituted with a natural orunnatural amino acid or peptidomimetic that does not contain a reactiveprimary amino group on a side chain, such as, e.g., Gly, Ser, Arg, Asn,Gln, Asp, Glu or citrulline (Cit). In a particular embodiment, Lys4and/or Lys10 are replaced with Arg. In another embodiment, Lys10 is notreplaced with Arg.

The disclosure contemplates use of hydrophilic or water soluble polymers(e.g., PEG molecules) that may vary in type (e.g., homopolymer orcopolymer; random, alternating or block copolymer; linear or branched;monodispersed or polydispersed), linkage (e.g., hydrolysable or stablelinkage such as, e.g., amide, imine, aminal, alkylene, or ester bond),conjugation site (e.g., at the N-terminus and/or C-terminus, preferablynot at any of the residues in the cyclized region of CNP (correspondingto residues 6-22 of CNP22)), and length (e.g., from about 0.2, 0.4 or0.6 kDa to about 2, 3, 4 or 5 kDa). The hydrophilic or water-solublepolymer can be conjugated to the CNP peptide by means of N-hydroxysuccinimide (NHS)- or aldehyde-based chemistry or other chemistry, as isknown in the art. Such CNP variants can be generated using, e.g.,wtCNP-22 (2.2 kDa), CNP-17 retaining only the cyclized region (residues6-22) of wtCNP22, CNP variants having an amino acid extension at theN-terminus and/or C-terminus of CNP22, or variants with amino acidsubstitutions, additions and/or deletions, for example, GANRR-CNP22(K4R)(SEQ ID NO: 36), GANPR-CNP22(K4R) (SEQ ID NO: 37), R-CNP22 (SEQ ID NO:40), R-CNP22(K4R) (SEQ ID NO: 41), ER-CNP22 (SEQ ID NO: 38) andER-CNP22(K4R) (SEQ ID NO: 39). In an embodiment, the PEG-CNP variantshaving a total mass characterized by the ranges described generallyherein, e.g., from about 2.6 kDa or 2.8 kDa to about 6 or 7 kDa, containa monodispersed, linear PEG (or PEO) group conjugated at the N-terminusand/or C-terminus via NHS- or aldehyde-based chemistry, or a two-arm orthree-arm branched PEG group conjugated at the N-terminus and/orC-terminus via NHS-based chemistry. The disclosure further contemplatesnegatively charged PEG-CNP variants designed for reduced renalclearance, including but not limited to carboxylated, sulfated andphosphorylated compounds.

In a related embodiment, the disclosure contemplates PEG-CNP conjugatescomprising NHS- or aldehyde-based PEG of the formula (CH₂CH₂O)_(n),wherein n is an integer from 12 to 50, and the PEG polymer is up toabout 2.5 kDa in molecular weight. In a specific embodiment, n is 12 or24. In an embodiment, the terminal hydroxyl group of the PEG polymer iscapped with a non-reactive group. In a particular embodiment, thecapping group is an alkyl group, e.g., a lower alkyl group such asmethyl.

In an additional embodiment, the PEG polymers or derivatives thereofhave a polymer number-average molecular weight in the range from about0.4 kDa to about 2.5 kDa or from about 0.6 kDa to about 1.5 kDa.

In a further embodiment, the wtCNP or CNP variant peptide is conjugatedto a moiety including, e.g., bisphosphonates, carbohydrates, hydrophobicacids (including fatty acids) or amino acid sequences. Such amino acidsequences include for example polyAsp or polyGlu useful inbone/cartilage targeting, or can be derived from bone proteins withelucidated bone-targeting domains or derivatives thereof, such as forexample fusion proteins or peptide sequences of osteopontin,osteocalcin, sialoprotein, etc. In embodiments described herein whereCNP22 or a variant thereof is attached to a bone- or cartilage-targetingmoiety, such a moiety is designed to promote getting the modified CNPpeptide to chondrocytes of bone growth plates, where the peptide canbind and activate NPR-B on the chondrocytes.

In another embodiment, the disclosure provides CNP variants with apeptide bond that is less susceptible to cleavage by peptidasesincluding NEP. The disclosure encompasses a CNP variant comprising atleast one modified residue at a site of endopeptidase cleavage. In oneembodiment, the Cys6-Phe7 peptide bond (—C(═O)—NH—) at an NEP cleavagesite in CNP can be replaced with anyone of the following peptide-bondisosteres:

—CH₂—NH—,

—C(═O)—N(R)—, where the amide group is alkylated with any of thefollowing R groups: methyl, ethyl, n-propyl, isopropyl, cyclopropyl,n-butyl, isobutyl, sec-butyl or tert-butyl,

—C(═O)—NH—CH₂—,

—CH₂—S—,

—CH₂—S(O)_(n)—, where n is 1 or 2,

—CH═CH—,

—C(═O)—CH₂—,

—CH(CN)—NH—,

—CH(OH)—CH₂—,

—O—C(═O)—NH—, and

—NHC(═O)NH—.

In another embodiment, Phe7 is substituted with its enantiomer D-Phe. Inyet another embodiment, D-enantiomers are introduced to one or more, upto all 22, positions within wtCNP-22. In a further embodiment, a betaamino acid such as 3-amino-2-phenylpropionic acid is substituted forPhe7, which increases the length of the backbone while reducing thelength of the side chain. In yet another embodiment, the disclosurecontemplates Cys analogs at the Cys6 position including but not limitedto homocysteine, penicillamine, 2-mercaptopropionic acid, and3-mercaptopropionic acid.

Even in the presence of a NEP-resistant bond between Cys6 and Phe7,other peptide bonds can be hydrolyzed by NEP, including Gly8-Leu9,Lys10-Leu11, Arg13-Ile14, Ser16-Met17 and Gly19-Leu20. Accordingly, thedisclosure encompasses CNP analogs containing peptide bond isosteres atmultiple locations in the backbone of the CNP analogs. In oneembodiment, CNP analogs or variants comprise modifications at more thanone peptidase cleavage site. In a further embodiment, such variantcomprises a CNP with substitutions at amino acid residues important inbinding to the NEP active site, thereby increasing resistance to NEPdegradation. One or more NEP-binding residues, including but not limitedto Gly8, Gly15, Ser18, Gly19 and/or Gly21, are replaced with larger-sizenatural or unnatural amino acid residues to reduce affinity to the NEPactive site. In yet another embodiment, one or more hydrophobic residuesessential in NEP recognition, including but not limited to Phe7, Leu9,Leu11, Ile14, Met17 and Leu20, are substituted with natural or unnaturalamino acids and/or peptidomimetics that decrease NEP binding. In yetanother embodiment, one to five of the first five amino acids of CNP canbe deleted or substituted with any other natural amino acids orunnatural amino acids or peptidomimetics, or one or more natural orunnatural amino acids or peptidomimetics can be added to any one or toall of the first five positions of CNP.

In a further embodiment, the CNP variants have a total masscharacterized by the ranges described generally herein, e.g., from about2.6 kDa or 2.8 kDa to about 6 or 7 kDa for increased resistance to NEP,and are represented by the formula:

-   (x)-Gly₁-Leu₂-Ser₃-(a)₄-Gly₅-(b)₆-(c)₇-(d)₈-(e)₉-(f)₁₀-(g)₁₁-Asp₁₂-Arg₁₃-(h)₁₄-Gly₁₅-Ser₁₆-(i)₁₇-Ser₁₈-Gly₁₉-(j)₂₀-Gly₂₁-Cys₂₂-(z)    (SEQ ID NO: 46), wherein:

(x) may be absent or may be selected from the group consisting ofsynthetic bone-targeting compounds such as, e.g., bisphosphonates; aminoacid sequences useful in bone or cartilage targeting such as, e.g.,polyAsp and polyGlu; amino acid sequences derived from bone proteinswith elucidated bone-targeting domains, such as, e.g., fusion proteinsor peptide sequences of osteopontin, osteocalcin, sialoprotein, etc.;polymeric or non-polymeric molecules that reduce renal clearance suchas, e.g., charged PEG molecules; and extensions comprising, e.g.,polymers (e.g., PEGs), carbohydrates, hydrophobic acids (including fattyacids), and/or amino acids, and wherein such amino acid extensions cancontain, e.g., from 1 to 31, or 1 to 35, or 5 to 35, or 10 to 35, or 15to 35 amino acid residues, and can be derived from NPPC, ANP, BNP, othernon-CNP (poly)peptides such as, e.g., serum albumin or IgG, or variantsof the aforementioned polypeptides having substitutions, additionsand/or deletions, or combinations thereof;

(z) may be absent or may be selected from the group consisting of aminoacid sequences useful in bone or cartilage targeting such as for examplepolyAsp and polyGlu, amino acid sequences from bone-targeting domains ofbone proteins such as, e.g., osteopontin, osteocalcin and sialoprotein,and amino acid sequences derived from non-CNP (poly)peptides such as,e.g., ANP or BNP;

(a) may be the wild type Lys at that position or may be replaced with aconservative amino acid substitution or a natural or unnatural aminoacid or peptidomimetic that does not have a reactive primary amine on aside chain, including but not limited to Arg, Gly, 6-hydroxy-norleucine,citrulline (Cit), Gln, Ser or Glu, wherein in one embodiment (a) is Arg;

(b) is selected from the group consisting of Cys and peptide-bondisosteres between Cys6 and Phe7 such as, e.g., Cys-CH₂—NH;

(c) is selected from the group consisting of L-Phe; D-Phe;3-amino-2-phenylpropionic acid; N-alkylated derivatives of Phe, whereinthe N-alkyl group is methyl, ethyl, n-propyl, isopropyl, cyclopropyl,n-butyl, isobutyl, sec-butyl or tert-butyl; and Phe analogs, wherein oneor more ortho-, meta-, and/or para-positions of the benzene ring of thePhe analog are substituted with one or more substituents selected fromthe group consisting of halogen, hydroxyl, cyano, straight or branchedC₁₋₆ alkyl, straight or branched C₁₋₆ alkoxy, straight or branchedhalo-C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl, heterocyclyl, C₆₋₁₄ aryl andheteroaryl (examples include, but are not limited to, tyrosine,3-chlorophenylalanine, 2,3-chloro-phenylalanine,3-chloro-5-fluoro-phenylalanine,2-chloro-6-fluoro-3-methyl-phenylalanine), or wherein the benzene ringof the Phe analog can be replaced with another aryl group (non-limitingexamples include 1- and 2-naphthylalanine) or with a heteroaryl group(non-limiting examples include pyridylalanine, thienylalanine andfurylalanine);

(d) is selected from the group consisting of Gly, tert-butyl-Gly(tBu-Gly), Thr, Ser, Val and Asn;

(e) is selected from the group consisting of Leu, Ser, Thr andpeptide-bond isosteres such as, e.g., N-Me-Leu;

(f) may be the wild type Lys at that position or may be replaced with aconservative amino acid substitution or a natural or unnatural aminoacid or peptidomimetic that does not have a reactive primary amine on aside chain, including but not limited to Arg, Gly, 6-hydroxy-norleucine,citrulline (Cit), Gln, Ser or Glu, wherein in one embodiment (f) is notArg;

(g) is selected from the group consisting of Leu and peptide-bondisosteres such as, e.g., N-Me-Leu;

(h) is selected from the group consisting of Ile, tBu-Gly, andpeptide-bond isosteres such as, e.g., N-Me-Ile;

(i) is selected from the group consisting of Met, Val, Asn, beta-Cl-Ala,2-aminobutyric acid (Abu) and 2-amino-isobutyric acid (Aib); and

(j) is selected from the group consisting of Leu, norleucine (Nle),homoleucine (Hleu), Val, tert-butyl-Ala (tBu-Ala), Ser, Thr, Arg, andpeptide-bond isosteres such as, e.g., N-Me-Leu.

In one embodiment, the CNP variants comprise a modification at one ormore of positions 6, 7, 8, 9, 10, 11, 13, 14, 16, 17, 19 and/or 20, andmay optionally have modifications at any of the other positionsdisclosed herein.

In embodiments described herein where CNP22 or variants thereof can beattached to a hydrophobic acid, the CNP peptides can be attached to oneor hydrophobic acids. Non-limiting examples of hydrophobic acids includestraight-chain or branched, saturated or unsaturated C₅-C₁₂ carboxylicacids (e.g., pentanoic acid, heptanoic acid, etc.) and natural fattyacids. The hydrophobic acids can be attached to the N-terminus, theC-terminus, and/or the side chain of one or more amino acid residues. Inone embodiment, the hydrophobic acids are conjugated to the N-terminus.In an embodiment, conjugation of CNP22 or a variant thereof to ahydrophobic acid is designed, inter alia, to promote non-specificinteraction between the modified CNP peptide and serum albumin, therebyincreasing the size of the CNP peptide and protecting it from cleavageby proteases such as, e.g., NEP. The interaction between the hydrophobicacid-conjugated CNP peptide and albumin is designed to be not toostrong, so that the modified CNP peptide can diffuse through cartilage,get to chondrocytes of bone growth plates, and bind and activate NPR-B.

In a further embodiment, the disclosure provides CNP variants that invitro or in vivo stimulate the production of at least about 50%, 60%,70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the cGMP levelproduced under the same concentration of wtCNP22 (e.g., 1 uM), compriseat least one modified amino acid at position (b)₆, (c)₇ and/or (d)₈, andare represented by the general formula:

-   (x)-Gly₁-Leu₂-Ser₃-(a)₄-Gly₅-(b)₆-(c)₇-(d)₈-(e)₉-(f)₁₀-(g)₁₁-Asp₁₂-Arg₁₃-(h)₁₄-Gly₁₅-Ser₁₆-(i)₁₇-Ser₁₈-Gly₁₉-(j)₂₀-Gly₂₁-Cys₂₂-(z)    (SEQ ID NO: 47), wherein:

(x) may be absent or may be a peptide sequence containing one to fiveamino acids which is derived from a natriuretic polypeptide (e.g. NPPC,CNP, ANP or BNP) or a non-natriuretic polypeptide as described herein(e.g., HSA, IgG, a bone-targeting protein, etc.);

(z) may be absent or may be selected from the group consisting ofsynthetic bone-targeting compounds such as, e.g., bisphosphonates; aminoacid sequences useful in bone/cartilage targeting such as, e.g., polyAspand polyGlu; bone proteins with bone-targeting domains and derivativesthereof, such as fusion proteins or peptides sequences of osteopontin,osteocalcin, and sialoprotein; molecules that reduce renal clearance,such as, e.g., charged PEGs; and molecules that increase resistance ofCNP to NEP-mediated degradation, as described herein;

(a) may be the wild type Lys at that position or may be replaced with aconservative amino acid substitution or a natural or unnatural aminoacid or peptidomimetic that does not have a reactive primary amine on aside chain, including but not limited to Arg, Gly, 6-hydroxy-norleucine,citrulline (Cit), Gln, Ser or Glu, wherein in one embodiment (a) is Arg;

(b) may be Cys or descarboxy cysteine, or the (b)₆-(c)₇ peptide bond(—C(═O)—NH—) may be replaced with any one of the following peptide bondisosteres:

-   -   —CH₂—NH—,    -   —C(═O)—N(R)—, where the amide group is alkylated with any of the        following R groups: methyl, ethyl, n-propyl, isopropyl,        cyclopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl,    -   —C(═O)—NH—CH₂—,    -   —CH₂—S—,    -   —CH₂—S(O)_(n)—, where n is 1 or 2,    -   —CH₂—CH₂—,    -   —CH═CH—,    -   —C(═O)—CH₂—,    -   —CH(CN)—NH—,    -   —CH(OH)—CH₂—,    -   —O—C(═O)—NH—, or    -   —NHC(═O)NH—;

(c) is selected from the group consisting of L-Phe; D-Phe;3-amino-2-phenylpropionic acid; peptide bond isosteres of Phe such asN-alkylated derivatives of Phe wherein the N-alkyl group is selectedfrom the group consisting of methyl, ethyl, n-propyl, isopropyl,cyclopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl; and Pheanalogs, wherein one or more ortho-, meta-, and/or para-positions of thebenzene ring of the Phe analog are substituted with one or moresubstituents selected from the group consisting of halogen, hydroxyl,cyano, straight or branched C₁₋₆ alkyl, straight or branched C₁₋₆alkoxy, straight or branched halo-C₁₋6 alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₄aryl, heterocyclyl and heteroaryl (examples include, but are not limitedto, tyrosine, 3-chlorophenylalanine, 2,3-chloro-phenylalanine,3-chloro-5-fluoro-phenylalanine,2-chloro-6-fluoro-3-methyl-phenylalanine), or wherein the benzene ringof the Phe analog can be replaced with another aryl group (non-limitingexamples include 1- and 2-naphthylalanine) or with a heteroaryl group(non-limiting examples include pyridylalanine, thienylalanine andfurylalanine);

(d) is selected from the group consisting of Gly, tert-butyl-Gly(tBu-Gly), Val, Ser, Thr and Asn;

(e) is selected from the group consisting of Leu, Ser, Thr, andpeptide-bond isosteres such as, e.g., N-Me-Leu;

(f) is any natural or unnatural amino acid or peptidomimetic that doesnot have a reactive primary amino group on a side chain, including butnot limited to Arg, Gly, 6-hydroxy-norleucine, citrulline (Cit), Gln,Ser or Glu, wherein in one embodiment (f) is not Arg;

(g) is selected from the group consisting of Leu and peptide-bondisosteres such as, e.g., N-Me-Leu;

(h) is selected from the group consisting of Ile, tBu-Gly andpeptide-bond isosteres such as, e.g., N-Me-Ile;

(i) is selected from the group consisting of Met, Val, Asn, beta-Cl-Ala,2-aminobutyric acid (Abu) and 2-amino-isobutyric acid (Aib); and

(j) is selected from the group consisting of Leu, norleucine (Nle),homoleucine (Hleu), Val, tert-butyl-Ala (tBu-Ala), Ser, Thr, Arg, andpeptide-bond isosteres such as, e.g., N-Me-Leu.

In a further embodiment, the disclosure encompasses CNP variants that invitro or in vivo stimulate the production of at least about 50%, 60%,70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the cGMP levelproduced under the same concentration of wtCNP22 (e.g., 1 uM), have atotal mass characterized by the ranges described generally herein, e.g.,from about 2.6 kDa or 2.8 kDa to about 6 or 7 kDa for increasedresistance to NEP degradation, and are represented by the generalformula:

-   (x)-(y)-Cys₆-Phe₇-Gly₈-Leu₉-(h)₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂-(z)    (SEQ ID NO: 48), wherein:

(x) is a synthetic or natural polymeric group, or a combination thereof,wherein a non-limiting example of a synthetic polymeric group ispolyethylene glycol (PEG), and a non-limiting example of a naturalpolymeric group is an amino acid sequence containing from 1 to 35 aminoacids and derived from NPPC or variants thereof with substitutionsand/or deletions, ANP, BNP, or other non-CNP (poly)peptides such as,e.g., serum albumin, IgG, histidine-rich glycoproteins, fibronectin,fibrinogen, zinc finger-containing polypeptides, osteocrin or FGF2;

(y) may be absent or may be one or more amino acids fromGly₁-Leu₂-Ser₃-Lys₄-Gly₅ (corresponding to positions 1 to 5 of CNP22)(SEQ ID NO: 1) and/or substitutions at one or more of those positionsusing natural or unnatural amino acids (e.g., K4R substitution);

(h) may be the wild-type Lys at that position or may be replaced with aconservative amino acid substitution or a natural or unnatural aminoacid or peptidomimetic that does not have a reactive primary amine on aside chain, including but not limited to Arg, Gly, 6-hydroxy-norleucine,citrulline (Cit), Gln, Ser or Glu, wherein in one embodiment (h) is notArg; and

(z) may be absent or may be a synthetic or natural polymeric group, or acombination thereof, wherein a non-limiting example of a syntheticpolymeric group is PEG, and a non-limiting example of a naturalpolymeric group is an amino acid sequence derived from a natriureticpolypeptide (e.g., NPPC, CNP, ANP or BNP) or non-natriuretic polypeptide(e.g., serum albumin or IgG).

In an embodiment, (x), (y) and (z) together contain from about 10 toabout 40, or from about 15 to about 35 amino acids. In anotherembodiment, (x) is an amino acid sequence comprising from 1 to 40 aminoacids, or from 1 to 20 amino acids.

Further contemplated are CNP variants that in vitro or in vivo stimulatethe production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%,120%, 130%, 140% or 150% of the cGMP level produced under the sameconcentration of wtCNP22 (e.g., 1 uM), and comprise the sequence:

-   (y)-Cys₆-Phe₇-Gly₈-Leu₉-Lys₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂    (SEQ ID NO: 138), wherein:

(y) comprises one or more amino acids selected fromGly₁-Leu₂-Ser₃-Lys₄-Gly₅ (SEQ ID NO: 1) and/or substitutions at one ormore of those positions using natural or unnatural amino acids (e.g.,K4R substitution), and further comprises a hydrophilic or water solublepolymer of molecular weight from about 0.6 kDa to about 5 kDa. In anembodiment, the hydrophilic or water-soluble polymer is conjugated tothe N-terminus of such amino acid-extended CNP variant. In a particularembodiment, the hydrophilic or water-soluble polymer is PEG (or PEO).

In yet another embodiment, the disclosure provides CNP variants that invitro or in vivo stimulate the production of at least about 50%, 60%,70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the cGMP levelproduced under the same concentration of wtCNP22 (e.g. 1 uM), whereinthe CNP variants comprise an N-terminal and/or C-terminal peptideextension containing from 1 to 15 amino acids, and are conjugated to ahydrophilic or water soluble polymer. In an embodiment, the peptideextension contains from 5 to 10 amino acids. In a specific embodiment,the peptide extension contains 5 amino acids. In another specificembodiment, the hydrophilic or water-soluble polymer is PEG (or PEO).

In a still further embodiment, the CNP variants of the disclosure invitro or in vivo stimulate the production of at least about 50%, 60%,70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the cGMP levelproduced under the same concentration of wtCNP22 (e.g. 1 uM), andcomprise at least a 15 amino acid fragment derived from natriureticpeptide precursor C(NPPC), wherein the fragment is at least 70%homologous to a sequence from wild type NPPC containing the same numberof amino acid residues.

In still another embodiment, the CNP variants have a total masscharacterized by the ranges described generally herein, e.g., from about2.6 kDa or 2.8 kDa to about 6 or 7 kDa for increased NEP resistance, andare represented by the formula:

-   (x)-(b)₆-(c)₇-(d)₈-(e)₉-(f)₁₀-(g)₁₁-Asp₁₂-Arg₁₃-(h)₁₄-Gly₁₅-Ser₁₆-(i)₁₇-Ser₁₈-Gly₁₉-(j)₂₀-Gly₂₁-Cys₂₂-(z)    (SEQ ID NO: 49), wherein:

(x) may be absent (i.e., the N-terminus ends with an —NH₂ group) or maybe selected from the group consisting of a sequence of 1, 2, 3, 4 or 5amino acids from the peptide Gly₁-Leu₂-Ser₃-Lys₄-Gly₅ (SEQ ID NO: 1);amino acid sequences useful in bone/cartilage targeting such as forexample polyAsp or polyGlu; bone-targeting domains from bone proteinssuch as for example osteopontin, osteocalcin or sialoprotein; moleculesthat reduce renal clearance such as hydrophilic or water-solublepolymers, including but not limited to charged PEG molecules; andmoieties comprising PEG, carbohydrates, hydrophobic acids, amino acids,or combinations thereof, wherein such moieties can be amino acidextensions including but not limited to amino acid sequences derivedfrom NPPC or non-CNP (poly)peptides such as, e.g., BNP, ANP, serumalbumin or IgG;

(z) may be absent or may be selected from the group consisting of aminoacid sequences useful in bone/cartilage targeting such as for examplepolyAsp or polyGlu; amino acid sequences derived from bone-targetingproteins, such as for example osteopontin, osteocalcin or sialoprotein;and amino acid sequences derived from NPPC or non-CNP (poly)peptides, asdescribed herein;

(b) is selected from the group consisting of Cys and peptide bondisosteres between Cys6 and Phe7 such as, e.g., Cys-CH₂—NH;

(c) is selected from the group consisting of L-Phe; D-Phe;3-amino-2-phenylpropionic acid; N-alkylated derivatives of Phe, whereinthe N-alkyl group is methyl, ethyl, n-propyl, isopropyl, cyclopropyl,n-butyl, isobutyl, sec-butyl or tert-butyl; and Phe analogs, wherein oneor more ortho-, meta-, and/or para-positions of the benzene ring of thePhe analog are substituted with one or more substituents selected fromthe group consisting of halogen, hydroxyl, cyano, straight or branchedC₁₋₆ alkyl, straight or branched C₁₋₆ alkoxy, straight or branchedhalo-C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₄ aryl, heterocyclyl andheteroaryl (examples include, but are not limited to, tyrosine,3-chlorophenylalanine, 2,3-chloro-phenylalanine,3-chloro-5-fluoro-phenylalanine,2-chloro-6-fluoro-3-methyl-phenylalanine), or wherein the benzene ringof the Phe analog can be replaced with another aryl group (non-limitingexamples include 1- and 2-naphthylalanine) or with a heteroaryl group(non-limiting examples include pyridylalanine, thienylalanine andfurylalanine);

(d) is selected from the group consisting of Gly, tert-butyl-Gly(tBu-Gly), Val, Ser, Thr and Asn;

(e) is selected from the group consisting of Leu, Ser, Thr, andpeptide-bond isosteres such as, e.g., N-Me-Leu;

(f) may be the wild type Lys at that position or may be replaced with aconservative amino acid substitution or a natural or unnatural aminoacid or peptidomimetic that does not have a reactive primary amine on aside chain, including but not limited to Arg, Gly, 6-hydroxy-norleucine,citrulline (Cit), Gln, Ser or Glu, wherein in one embodiment (f) is notArg;

(g) is selected from the group consisting of Leu and peptide-bondisosteres such as, e.g., N-Me-Leu;

(h) is selected from the group consisting of Ile, tBu-Gly andpeptide-bond isosteres such as, e.g., N-Me-Ile;

(i) is selected from the group consisting of Met, Val, Asn, beta-Cl-Ala,2-aminobutyric acid (Abu) and 2-amino-isobutyric acid (Aib); and

(j) is selected from the group consisting of Leu, norleucine (Nle),homoleucine (Hleu), Val, tert-butyl-Ala (tBu-Ala), Ser, Thr, Arg, andpeptide-bond isosteres such as, e.g., N-Me-Leu.

In a further embodiment, the CNP variants have a total masscharacterized by the ranges described generally herein, e.g., from about2.6 kDa or 2.8 kDa to about 6 or 7 kDa for increased NEP resistance, andare represented by the formula:

-   (x)-Gly₁-Leu₂-Ser₃-(a)₄-Gly₅-(b)₆-(c)₇-(d)₈-(e)₉-(f)₁₀-(g)₁₁-Asp₁₂-Arg₁₃-(h)₁₄-(i)₁₅-Ser₁₆-(j)₁₇-Ser₁₈-Gly₁₉-(k)₂₀-Gly₂₁-Cys₂₂-(z)    (SEQ ID NO: 50), wherein:

(x) and (z) independently may be absent or may be selected from thegroup consisting of synthetic bone-targeting compounds such as, e.g.,bisphosphonates; amino acid sequences useful in bone/cartilage targetingsuch as for example polyAsp or polyGlu; amino acid sequences derivedfrom bone-targeting domains of bone proteins and derivatives thereof,such as for example fusion proteins or peptide sequences of osteopontin,osteocalcin, sialoprotein, etc.; moieties that reduce renal clearance,including but not limited to hydrophilic or water-soluble polymers suchas, e.g., charged PEG molecules; and moieties comprising, e.g.,hydrophilic polymers (e.g., PEG), carbohydrates, hydrophobic acids,and/or amino acids;

(a) may be the wild type Lys at that position or may be replaced with aconservative amino acid substitution or a natural or unnatural aminoacid or peptidomimetic that does not have a reactive primary amine on aside chain, including but not limited to Arg, Gly, 6-hydroxy-norleucine,citrulline (Cit), Gln, Ser or Glu, wherein in one embodiment (a) is Arg;

(b) is selected from the group consisting of Cys and peptide bondisosteres between Cys6 and Phe7 such as, e.g., Cys-CH₂—NH;

(c) is selected from the group consisting of L-Phe; D-Phe;3-amino-2-phenylpropionic acid; N-alkylated derivatives of Phe, whereinthe N-alkyl group is methyl, ethyl, n-propyl, isopropyl, cyclopropyl,n-butyl, isobutyl, sec-butyl or tert-butyl; and Phe analogs, wherein oneor more ortho-, meta-, and/or para-positions of the benzene ring of thePhe analog are substituted with one or more substituents selected fromthe group consisting of halogen, hydroxyl, cyano, straight or branchedC₁₋₆ alkyl, straight or branched C₁₋₆ alkoxy, straight or branchedhalo-C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₄ aryl, heterocyclyl andheteroaryl (examples include, but are not limited to, tyrosine,3-chlorophenylalanine, 2,3-chloro-phenylalanine,3-chloro-5-fluoro-phenylalanine,2-chloro-6-fluoro-3-methyl-phenylalanine), or wherein the benzene ringof the Phe analog can be replaced with another aryl group (non-limitingexamples include 1- and 2-naphthylalanine) or with a heteroaryl group(non-limiting examples include pyridylalanine, thienylalanine andfurylalanine);

(d) is selected from the group consisting of Gly, tert-butyl-Gly, Thr,Ser, Val and Asn;

(e) is selected from the group consisting of Leu, Ser, Thr, and peptidebond isosteres such as, e.g., N-Me-Leu;

(f) may be the wild type Lys at that position or may be replaced with aconservative amino acid substitution or a natural or unnatural aminoacid or peptidomimetic that does not have a reactive primary amine on aside chain, including but not limited to Arg, Gly, 6-hydroxy-norleucine,citrulline (Cit), Gln, Ser or Glu, wherein in one embodiment (f) is notArg;

(g) is selected from the group consisting of Leu, Asn, and peptide bondisosteres such as, e.g., N-Me-Leu;

(h) is selected from the group consisting of Ile, tert-butyl-Gly(tBu-Gly), Asn, and peptide bond isosteres such as, e.g., N-Me-Ile;

(i) is selected from the group consisting of Gly, Arg, Ser and Asn;

(j) is selected from the group consisting of Met, Val, Asn, beta-Cl-Ala,2-aminobutyric acid (Abu) and 2-amino-isobutyric acid (Aib); and

(k) is selected from the group consisting of Leu, norleucine (Nle),homoleucine (Hleu), Val, tert-butyl-Ala (tBu-Ala), Arg, Thr, Ser, andpeptide bond isosteres such as, e.g., N-Me-Leu.

In a further embodiment, the CNP variants can have amino acidsubstitution(s) at one or more of any of positions 1 to 22 of CNP22. Inone embodiment, Gly1 is substituted with Arg or Glu. In anotherembodiment, Lys4 is replaced with Arg. In still another embodiment, Gly5is substituted with Arg, Gln or Ser. In yet another embodiment, Gly15 issubstituted with Ser, Asn, Arg or Cit. In a further embodiment, Gly19 issubstituted with Ser, Arg or Asn. In yet another embodiment, Gly21 issubstituted with Ser, Thr, or Arg.

In one embodiment, the CNP variant is selected from the group consistingof GLSKGC(CH₂NH)FGLKLDRIGSMSGLGC (formed using descarboxy-Cys) (SEQ IDNO: 56), GLSKGC-(N-Me-Phe)-GLKLDRIGSMSGLGC (SEQ ID NO: 57),GLSKGC-(D-Phe)-GLKLDRIGSMSGLGC(SEQ ID NO:136),GLSKGCF-(tBuG)-LKLDRIGSMSGLGC (SEQ ID NO: 58),GLSKGC-(3-Cl-Phe)-GLKLDRIGSMSGLGC (SEQ ID NO:137), andGLSKGC-[NHCH₂CH(Ph)CO]-GLKLDRIGSMSGLGC (formed using3-amino-2-phenylpropionic acid) (SEQ ID NO: 59). In a furtherembodiment, a disulfide bond exists between Cys6, descarboxy-Cys oranother sulfhydryl-containing cysteine analog at the Cys6 position, andCys22 of any CNP variant described herein.

In another embodiment, the CNP variants contain an amino acid extensionat the N-terminus and/or C-terminus of CNP22 or CNP17, including but notlimited to:

(SEQ ID NO: 4) DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC(CNP-53); (SEQ ID NO: 60) QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37,Analog BL); (SEQ ID NO: 61) AAWARLLQEHPNAGLSKGCFGLKLDRIGSMSGLGC (AnalogCA); (SEQ ID NO: 62) AAWARLLQEHPNARGLSKGCFGLKLDRIGSMSGLGC (Analog CB);(SEQ ID NO: 63) DLRVDTKSRAAWARGLSKGCFGLKLDRIGSMSGLGC (Analog CC); (SEQID NO: 40) RGLSKGCFGLKLDRIGSMSGLGC; (SEQ ID NO: 38)ERGLSKGCFGLKLDRIGSMSGLGC; (SEQ ID NO: 64) GANQQGLSKGCFGLKLDRIGSMSGLGC;(SEQ ID NO: 65) GANRRGLSKGCFGLKLDRIGSMSGLGC; (SEQ ID NO: 66)GANPRGLSKGCFGLKLDRIGSMSGLGC; (SEQ ID NO: 67)GANSSGLSKGCFGLKLDRIGSMSGLGC; (SEQ ID NO: 144)GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC (sometimes designated“CNP27-HSA” or “HSA-CNP27” in the Examples and figures); (SEQ ID NO: 68)SPKMVQGSG-CNP17-KVLRRH (Analog CD) (CNP17 having N-terminal andC-terminal tails derived from BNP).

In a further embodiment, the CNP variants have a K4R substitution atposition 4 of CNP22. Non-limiting examples of CNP(K4R) variants include:

((SEQ ID NO: 36) GANRRGLSRGCFGLKLDRIGSMSGLGC (Analog AY); (SEQ ID NO:37) GANPRGLSRGCFGLKLDRIGSMSGLGC (Analog CI); (SEQ ID NO: 41)RGLSRGCFGLKLDRIGSMSGLGC (Analog AZ); (SEQ ID NO: 39)ERGLSRGCFGLKLDRIGSMSGLGC (Analog BA); (SEQ ID NO: 69)GANQQGLSRGCFGLKLDRIGSMSGLGC (Analog CH); and (SEQ ID NO: 70)GANSSGLSRGCFGLKLDRIGSMSGLGC (Analog CG).

In one embodiment, CNP variants having a PEG (or PEO) moiety and anamino acid extension at the N-terminus contain arginine at the positionimmediately preceding the position corresponding to Gly1 of CNP22. SuchPEGylated CNP variants are designed for increased resistance to NEPdegradation, reduced binding to serum albumin, and enhanced CNPfunctional activity (e.g., activation of cGMP signaling). Non-limitingexamples of PEGylated CNP variants include PEO24-GANRR-CNP22(K4R) (SEQID NO: 36), PEO12-GANRR-CNP22(K4R) (SEQ ID NO: 36),PEO24-GANRR-CNP22(SEQ ID NO: 65), PEO12-GANRR-CNP22(SEQ ID NO: 65),PEO24-GANPR-CNP22(K4R) (SEQ ID NO: 37), PEO12-GANPR-CNP22(K4R) (SEQ IDNO: 37), PEO24-GANPR-CNP22(SEQ ID NO: 37), PEO12-GANPR-CNP22(SEQ ID NO:66), PEO24-GANQQ-CNP22(SEQ ID NO: 64), PEO12-GANQQ-CNP22(SEQ ID NO: 64),PEO24-ER-CNP22(K4R) (SEQ ID NO: 39), PEO12-ER-CNP22(K4R) (SEQ ID NO:39), PEO24-ER-CNP22(SEQ ID NO: 38), PEO12-ER-CNP22(SEQ ID NO: 38),PEO24-R-CNP22(K4R) (SEQ ID NO: 41), PEO12-R-CNP22(K4R) (SEQ ID NO: 41),PEO24-R-CNP22(SEQ ID NO: 40), and PEO12-R-CNP22(SEQ ID NO: 40), whereinPEO24 is a monodispersed 1.2 kDa PEG polymer and PEO12 is amonodispersed 0.6 kDa PEG polymer. In an embodiment, the PEG (or PEO)polymer is attached to the N-terminus of the CNP variants.

Additional CNP variants include, but are not limited to, derivatives ofCNP37 having mutation(s) at the furin cleavage site (underlined),designed to improve in vivo resistance to the furin protease, and/orhaving glycine (underlined) preceding glutamine, designed to preventpyroglutamine formation, including but not limited to:

(SEQ ID NO: 71) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (An. CS); (SEQ IDNO: 72) GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (An. CT); (SEQ ID NO: 73)GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (An. CU); (SEQ ID NO: 74)GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (An. CW); (SEQ ID NO: 75)GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP37, An. DB); (SEQ ID NO:145) PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP37).

In another embodiment, the CNP variants are chimera comprising CNP22 andan N-terminal peptide fragment, including but not limited to:

(SEQ ID NO: 76) GHHSHEQHPHGANQQGLSKGCFGLKLDRIGSMSGLGC (Analog CQ)(histidine-rich glycoprotein (HRGP) fragment- CNP22 chimera);(SEQ ID NO: 77) GAHHPHEHDTHGANQQGLSKGCFGLKLDRIGSMSGLGC (Analog CR)(HRGP fragment-CNP22 chimera); (SEQ ID NO: 78)GHHSHEQHPHGANPRGLSKGCFGLKLDRIGSMSGLGC (Analog CX)(HRGP fragment-CNP22 chimera); (SEQ ID NO: 79)GQPREPQVYTLPPSGLSKGCFGLKLDRIGSMSGLGC (Analog CF)(IgG₁(F_(c)) fragment-CNP22 chimera); (SEQ ID NO: 80)GQHKDDNPNLPRGANPRGLSKGCFGLKLDRIGSMSGLGC (AnalogCY) (human serum albumin (HSA) fragment-CNP22 chimera); (SEQ ID NO: 81)GERAFKAWAVARLSQGLSKGCFGLKLDRIGSMSGLGC (Analog CE)(HSA fragment-CNP22 chimera); (SEQ ID NO: 82)FGIPMDRIGRNPRGLSKGCFGLKLDRIGSMSGLGC (Analog CZ)(osteocrin “NPR C inhibitor” fragment-CNP22 chimera); and(SEQ ID NO: 83) GKRTGQYKLGSKTGPGPKGLSKGCFGLKLDRIGSMSGLGC(Analog DA) (FGF2 “heparin-binding domain” fragment-CNP22 chimera).

In a further embodiment, the CNP variants are chimera comprising anN-terminal peptide fragment and CNP22 in which arginine is substitutedfor Lys4 of CNP22 (“CNP22(K4R)”), including but not limited to:

(SEQ ID NO: 84) GQPREPQVYTGANQQGLSRGCFGLKLDRIGSMSGLGC (Analog CK)(IgG₁(F_(c)) fragment-CNP22(K4R) chimera); (SEQ ID NO: 85GVPQVSTSTGANQQGLSRGCFGLKLDRIGSMSGLGC (Analog CL)(HSA fragment-CNP22(K4R) chimera) (SEQ ID NO: 86)GQPSSSSQSTGANQQGLSRGCFGLKLDRIGSMSGLGC(Analog CM) (fibronectin fragment-CNP22(K4R) chimera); (SEQ ID NO: 87)GQTHSSGTQSGANQQGLSRGCFGLKLDRIGSMSGLGC(Analog CN) (fibrinogen fragment-CNP22(K4R) chimera); (SEQ ID NO: 88)GSTGQWHSESGANQQGLSRGCFGLKLDRIGSMSGLGC(Analog CO) (fibrinogen fragment-CNP22(K4R) chimera); and(SEQ ID NO: 89) GSSSSSSSSSGANQQGLSRGCFGLKLDRIGSMSGLGC(Analog CP) (zinc finger fragment-CNP22(K4R) chimera).

In yet another embodiment, the CNP variants are chimera, or fusionproteins, comprising a CNP peptide or variant, and a cleavable peptideor protein, or peptide tag. Exemplary cleavable proteins or peptidesinclude, but are not limited to, histidine (e.g., hexa-His) tags; TAF12:human transcription factor TAF12; KSI: ketosteroid isomerase; MBP:maltose-binding protein; β-Gal: β-galactosidase; GST:glutathione-S-transferase; Trx: thioredoxin; CBD: chitin binding domain;BMPM: BMP-2 mutation, SUMO, CAT, TrpE, staphylococcal protein A,streptococcal proteins, starch-binding protein, cellulose-binding domainof endoglucanase A, cellulose-binding domain of exoglucanase Cex,biotin-binding domain, recA, Flag, c-Myc, poly(His), poly(Arg),poly(Asp), poly(Gln), poly(Phe), poly(Cys), green fluorescent protein,red fluorescent protein, yellow fluorescent protein, cyan fluorescentprotein, biotin, avidin, streptavidin, antibody epitopes, and fragmentsthereof.

In yet another embodiment, the CNP variant may be a monomer or a dimer.In a related embodiment the monomers of dimeric CNP variants can beattached N-terminus to N-terminus via a linker or no linker, N-terminusto C-terminus via a linker or no linker, or C-terminus to C-terminus viaa linker or no linker.

Chimera comprising an IgG fragment and CNP22 or a variant thereof aredesigned for, inter alia, increased resistance to NEP degradation andreduced binding to serum albumin. CNP chimera comprising a surfacefragment of HSA are designed for, inter alia, reduced immunogenicity andreduced binding to serum albumin. HRGP-CNP22 and HRGP-CNP22(K4R) chimeracontaining a cationic, histidine-rich, non-lysine, non-arginine sequenceat the N-terminus are designed for, inter alia, increased stability toproteases. Chimera containing an osteocrin fragment are designed torelease, upon protease (e.g., furin) cleavage, the osteocrin fragment atbone growth plates, where the fragment would inhibit the clearancereceptor NPR-C. With respect to chimera comprising an FGF2heparin-binding fragment, heparin binding to the fragment is designed toprotect the chimera from degradation, thereby providing a longer serumhalf-life. Chimera containing a fibronectin, fibrinogen, or zinc-fingerfragment are designed for reduced binding to serum albumin, among otheradvantageous features.

Not intending to be bound by theory, a CNP variant of molecular weightfrom about 2.6 kDa or 2.8 kDa to about 6 or 7 kDa which has increasedresistance to NEP degradation and has similar or improved functionality(e.g., binding to NPR-B and stimulation of cGMP signaling) as comparedto wtCNP22, may be more effective if it does not bind tightly to plasmaproteins such as serum albumin. A CNP variant that does not bind tightlyto plasma proteins (e.g., serum albumin) may be more effective indiffusing through cartilage, getting to chondrocytes of bone growthplates, and binding to and activating NPR-B for cGMP signaling. In oneembodiment, CNP variants designed for reduced binding to plasma proteins(e.g., serum albumin) are chimeras comprising CNP22 or a variant thereofand a peptide fragment from IgG. In another embodiment, CNP variantsdesigned for reduced binding to plasma proteins are chimeras comprisingCNP22 or CNP22(K4R) and a fragment from a polypeptide (e.g., IgG, HSA,fibronectin, fibrinogen, a zinc finger-containing polypeptide, etc.). Inyet another embodiment, CNP variants designed for reduced binding toplasma proteins comprise CNP22 or a variant thereof conjugated to ahydrophilic or water-soluble polymer. In one embodiment, the hydrophilicor water-soluble polymer is PEG (or PEO). In another embodiment, thehydrophilic or water-soluble polymer (e.g., PEG) is functionalized withone or more functional groups that impart a negative charge to thepolymer under physiological conditions, such as, e.g, carboxyl, sulfateor phosphate groups, or a combination thereof.

In any of the embodiments disclosed herein, the CNP variants may havesubstantially the same or better biological activity than wild-typeCNP22. For example, the CNP variants may retain at least 50%, 60%, 70%,80%, 90%, 95% or more of the activity of wild-type CNP22, or may havegreater activity than CNP22, e.g., with respect to interaction withNPR-B (GC-B) to stimulate the generation of cGMP. Alternatively, or inaddition, the CNP variants may retain at least 50%, 60%, 70%, 80%, 90%,95% or more of the activity of wild-type CNP22, or may have greateractivity than CNP22, with respect to regulating endochondral bone growthand chondrocyte activity, including but not limited to chondrocyteproliferation, chondrocyte differentiation, inhibition of the mitogenactivated protein (MAP) kinase/MEK (Raf-1) kinase signaling pathway, andpromoting endochondral ossification. In any of the embodiments describedherein, the CNP variants may comprise an amino acid sequence that is atleast 40%, 50%, 60%, 70%, 80%, 90%, 95% or more identical or homologousto amino acids 6-22 or 1-22 of wild-type CNP22.

In a further embodiment, the disclosure provides variants of CNP22having less affinity to the NPR-C clearance receptor while retaining theability to bind and activate NPR-B. The present disclosure encompassesvariants that were, or can be, generated from a homology-basedstructural model of the NPR-B/CNP complex as described in the DetailedDescription. In another embodiment, the CNP variants havesubstitution(s) at one or more Gly sites at positions 1, 5, 8, 15, 19and 21, to reduce conformational flexibility, which may increase theirspecificity for binding to NPR-B over NPR-C. Variants of CNP havingpotentially reduced affinity to the NPR-C include but are not limited tothose having one or more of the following substitutions: G1R, G1E, G5R,G5Q, G5S, F7Y, G8T, G8S, G8V, G8N, L9S, L9T, K10Cit, K10Q, K10S, I14N,G15R, G15S, G15N, G15Cit, S16Q, M17V, M17N, G19S, G19R, G19N, L20V,L20R, L20T, L20S, G21S, G21T and G21R.

In yet another embodiment, the CNP variants have a modification and/orsubstitution at one or more of positions 5, 7, 8, 9, 10, 14, 15, 16, 17,19, 20 and 21, and may optionally have modifications and/orsubstitutions at any of the other positions disclosed herein. In afurther embodiment, the CNP variants can optionally have conjugation(s)or extension(s), e.g., at the N- and/or C-terminus to facilitatebone/cartilage targeting, reduce renal clearance, and/or increaseresistance to NEP degradation. Such conjugation(s) or extension(s) cancomprise molecules or sequences formed or derived from, e.g., polyAsp,polyGlu, bone- or cartilage-targeting peptides, osteopontin,osteocalcin, sialoprotein, PEGs, carbohydrates, hydrophobic acids, NPPCor non-CNP (poly)peptides, or combinations thereof.

In still another embodiment, the CNP variants are prepared by standardsolid-phase peptide synthesis methods with natural or unnatural aminoacid(s) or peptidomimetic(s) being substituted and/or added whereappropriate. In another embodiment, the CNP variants are produced byrecombinant synthesis processes, e.g., via fusion proteins containing atag or carrier protein, wherein use of the tag or carrier proteinfacilitates, e.g., detection, isolation and/or purification of thefusion protein, and selective chemical or proteolytic cleavage of thetag or carrier protein from the fusion protein provides the target CNPvariant. In a further embodiment, PEGylation of the CNP variants occursfollowing, or part of, chemical or biological synthesis with theconjugation reaction being performed by NHS- or aldehyde-based chemistryor other chemistry known in the art. In another embodiment, the CNPvariants comprise a disulfide bond. In a related embodiment, thedisulfide bond forms a cyclic peptide. In a particular embodiment, thedisulfide bond is formed between cysteine residues at positionscorresponding to positions 6 and 22 of CNP22.

It is further contemplated that the CNP variants can be conjugated to ahydrophobic polymeric or non-polymeric moiety, such as, e.g., heptanoicacid, pentanoic acid, or fatty acids. The hydrophobic moiety can beconjugated to the side chain of an amino acid residue, including but notlimited to a lysine, a serine, a cysteine or a threonine, or can beattached to the N-terminus and/or C-terminus of the CNP variant.

In an embodiment, the CNP variants as described herein have a pI in therange from about 8 to about 10.5 or from about 8.5 to about 10.

In a further embodiment, the disclosure provides a pharmaceuticalcomposition comprising a CNP variant, optionally another biologicallyactive agent, and optionally a pharmaceutically acceptable excipient,carrier or diluent. In some embodiments, the compositions are sterilepharmaceutical compositions suitable for parenteral injection. In someembodiments, the compositions comprise substantially pure CNP variant,e.g. at least about 90% or 95% pure. In some embodiments, thecompositions contain less than about 5%, 4%, 3%, 2%, 1% or 0.5%contaminants, such as other human proteins, porcine proteins, or CNP53or fragments thereof (other than the desired CNP variant). In certainembodiments, the sterile composition is administered to a subject fortreating or preventing any of the CNP-responsive conditions or disordersdisclosed herein.

CNP variants of the disclosure advantageously retain CNP activity andexhibit increased serum half-life. Retention of CNP activity can beshown, for example, as retention of desired in vivo biological effect,or retention of at least about 50%, 60%, 70%, 80%, 90%, 95% or at leastabout 100% of the cGMP stimulating activity of CNP22, under the sameconcentration (e.g., 1 uM of CNP peptide or greater than the ED80). Insome embodiments, CNP variants exhibit at least about 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold,35-fold or 40-fold increase in serum half-life compared to CNP22.

In a related embodiment, the CNP variants described herein haveincreased NEP resistance and exhibit increased half-life compared towild-type CNP22. In one embodiment, the half-life of the CNP variants isincreased by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99% or about 100% compared to wild-type CNP22.

In certain embodiments, the CNP variants described herein increase cGMPproduction in vitro, increase cGMP production in vivo, increase in vivothe level of one or more biomarkers associated with cartilage or boneformation or growth, increase resistance to NEP cleavage in vitro,increase plasma or serum half-life in vivo, increase bioavailability invivo, or increase the length of particular bones in vivo, or effectcombinations of such increases, by about 1.5-fold, about 2-fold, about2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, orabout 5-fold or more compared to wild-type CNP22.

In yet another embodiment, the disclosure provides methods of treatingconditions or disorders responsive to CNP, comprising administering atherapeutically effective amount of a CNP variant or a compositioncomprising the same to a subject in need thereof. In one embodiment,disorders responsive to CNP are disorders of bone growth, including butnot limited to skeletal dysplasias and inherited skeletal malformationssuch as disorders associated with fibroblast growth factor receptor 3(FGFR-3) mutations. In a specific embodiment, the disorder associatedwith FGFR-3 mutation(s) is achondroplasia. In another embodiment, thedisorders responsive to CNP are disorders associated with vascularsmooth muscle cells and tissues. In a further embodiment, the CNPvariants are useful for increasing the size of the growth plate of abone (e.g., a limb bone). In another embodiment, the CNP variants areuseful for elongating a bone or increasing long bone growth. In stillanother embodiment, the CNP variants are useful for enhancing matrixproduction, proliferation and differentiation of chondrocytes.

In certain embodiments, the CNP variants described herein areadministered at a dose in the range from about 5 or 10 nmol/kg to about300 nmol/kg, or from about 20 nmol/kg to about 200 nmol/kg. In someembodiments, the CNP variants are administered at a dose of about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,350, 400, 450, 500, 750, 1000, 1250, 1500, 1750 or 2000 nmol/kg or otherdose deemed appropriate by the treating physician. In other embodiments,the CNP variants are administered at a dose of about 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or 800 ug/kg,or about 1 mg/kg, 1.25 mg/kg, 1.5 mg/kg, 2 mg/kg or other dose deemedappropriate by the treating physician. The doses of CNP variantsdescribed herein can be administered according to the dosingfrequency/frequency of administration described herein, includingwithout limitation daily, 2 or 3 times per week, weekly, every 2 weeks,every 3 weeks, monthly, etc.

In another embodiment, the CNP variants are administered in a singletreatment or in multiple doses. The multiple doses may be administereddaily, or in multiple doses over the course of treatment. In certainembodiments, it is contemplated that the CNP variant is administered, ina single dose or in multiple doses, daily, every other day, every 3days, 2 times per week, 3 times per week, weekly, bi-weekly, every 3weeks, monthly, every 6 weeks, every 2 months, every 3 months or asdeemed appropriate by a treating physician.

In certain embodiments, administration of the CNP variant is adjusted toallow for periods of growth (e.g., chondrogenesis), followed by arecovery period (e.g., osteogenesis). For example, the CNP variant maybe administered subcutaneously, intravenously, or by another mode dailyor multiple times per week for a period of time, followed by a period ofno treatment, then the cycle is repeated. In some embodiments, theinitial period of treatment (e.g., administration of the CNP variantdaily or multiple times per week) is for 3 days, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks,11 weeks or 12 weeks. In a related embodiment, the period of notreatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. Incertain embodiments, the dosing regimen of the CNP variant is daily for3 days followed by 3 days off; or daily or multiple times per week for 1week followed by 3 days or 1 week off; or daily or multiple times perweek for 2 weeks followed by 1 or 2 weeks off; or daily or multipletimes per week for 3 weeks followed by 1, 2 or 3 weeks off; or daily ormultiple times per week for 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeksfollowed by 1, 2, 3 or 4 weeks off.

In additional embodiments, the disclosure provides a method of treatinga CNP-responsive condition or disorder, comprising administering a CNPpeptide or variant to a subject, and monitoring the level of at leastone bone- or cartilage-associated biomarker in the subject (e.g., in abiological sample from the subject), wherein an increase or decrease inthe level of the bone- or cartilage-associated biomarker indicates atherapeutic effect of the CNP peptide or variant on the subject. In someembodiments, when the level of a biomarker increases in association withbone or cartilage formation or growth, an increase in the level of thatbiomarker indicates a therapeutic effect of the CNP peptide or varianton the subject. In other embodiments, when the level of a biomarkerdecreases in association with bone or cartilage formation or growth, adecrease in the level of that biomarker indicates a therapeutic effectof the CNP peptide or variant on the subject.

In further embodiments, the therapeutic method further comprisesadjusting the amount (or dose) or frequency of administration of the CNPpeptide or variant, wherein:

-   (i) the amount (or dose) or frequency of administration of the CNP    peptide or variant is increased if the level of the at least one    bone- or cartilage-associated biomarker is below a target level,    where the level of the biomarker increases in association with bone    or cartilage formation or growth; or-   (ii) the amount (or dose) or frequency of administration of the CNP    peptide or variant is decreased if the level of the at least one    bone- or cartilage-associated biomarker is above a target level,    where the level of the biomarker increases in association with bone    or cartilage formation or growth; or-   (iii) the amount (or dose) or frequency of administration of the CNP    peptide or variant is increased if the level of the at least one    bone- or cartilage-associated biomarker is above a target level,    where the level of the biomarker decreases in association with bone    or cartilage formation or growth; or-   (iv) the amount (or dose) or frequency of administration of the CNP    peptide or variant is decreased if the level of the at least one    bone- or cartilage-associated biomarker is below a target level,    where the level of the biomarker decreases in association with bone    or cartilage formation or growth.    It is contemplated that the target level of a biomarker refers to    the level or range of levels of the biomarker that is associated    with therapeutic effect in the subject and/or beneficial effect in    alleviating or ameliorating symptoms of the disorder or condition.    In certain embodiments, a level of a biomarker above or below a    target level may be deleterious to the subject.

In other embodiments, the disclosure contemplates a method for assessingthe effect of administration of a CNP peptide or variant on bone orcartilage formation or growth. In one embodiment, the method providesfor assaying or measuring the level of at least one bone- orcartilage-associated biomarker in a subject that has been administered aCNP peptide or variant in order to assess the effect of the CNP peptideor variant on bone and cartilage formation and growth in vivo. In arelated embodiment, an increase in the level of the at least one bone-or cartilage-associated biomarker may indicate that administration of aCNP peptide or variant has a positive effect on bone or cartilageformation or growth and is a useful treatment for skeletal dysplasiasand other bone- or cartilage-related diseases or disorders associatedwith decreased CNP activity. Exemplary bone- or cartilage-associatedbiomarkers include, but are not limited to, CNP (e.g, endogenous levelof CNP-22 or CNP-53), cGMP, osteocalcin, proliferating cell nuclearantigen (PCNA), propeptides of type I procollagen (PINP) and fragmentsthereof, collagen type I and fragments thereof, propeptides of collagentype II and fragments thereof, collagen type II and fragments thereof,aggrecan chondroitin sulfate, and alkaline phosphatase.

In further embodiments, the disclosure contemplates a method forassessing the effect of a CNP peptide or variant on the level of atleast one bone- or cartilage-associated biomarker in a subject,comprising assaying or measuring the level of the bone- orcartilage-associated biomarker in a biological sample from a subjectthat has been administered a CNP peptide or variant. In someembodiments, the method further comprises administering the CNP peptideor variant to the subject before assaying or measuring the level of thebone- or cartilage-associated biomarker.

In certain embodiments, the at least one bone- or cartilage-associatedbiomarker is selected from the group consisting of CNP (e.g, endogenouslevel of CNP-22 or CNP-53), cGMP, propeptides of collagen type II andfragments thereof, collagen type II and fragments thereof, osteocalcin,proliferating cell nuclear antigen (PCNA), propeptides of type Iprocollagen (PINP) and fragments thereof, collagen type I and fragmentsthereof, aggrecan chondroitin sulfate, and alkaline phosphatase.

In some embodiments of methods (e.g., therapeutic, diagnostic and assaymethods) relating to bone- or cartilage-associated biomarkers, the CNPpeptide or variant is CNP-22, CNP-53, or any of the CNP peptides andvariants described herein. In certain embodiments of such methods, theCNP peptide or variant is not CNP-22 or CNP-53.

In other embodiments, the disclosure provides a method for recombinantproduction of a CNP variant, comprising culturing in a medium a hostcell comprising a polynucleotide encoding a CNP variant peptide linkedto a polynucleotide encoding a cleavable peptide or protein, underconditions that result in expression of a fusion polypeptide encoded bythe polynucleotides. In a related embodiment, the host cell istransformed with an expression vector comprising a polynucleotideencoding a CNP variant peptide linked to a polynucleotide encoding acleavable peptide or protein.

In one embodiment, the vector is a plasmid. In still another embodiment,the plasmid is selected from the group consisting of pET-21a, pJexpress,pET-31b, pET-15b, pET-32a, pET-41a, pMAL, pQE-30, pET-SUMO, pET-22b, andpTYB11.

In certain embodiments, the cleavable peptide or protein comprises apolypeptide that is selected from the group consisting of a histidinetag, human transcription factor TAF12, ketosteroid isomerase,maltose-binding protein, β-galactosidase, glutathione-S-transferase,thioredoxin, chitin binding domain, and BMP-2 mutation, or fragmentsthereof.

In a related embodiment, the cleavable peptide or protein is cleaved bya cleaving agent. In some embodiments, the cleaving agent is selectedfrom the group consisting of formic acid, cyanogen bromide (CNBr),hydroxylamine, protein self cleavage, Factor Xa, enterokinase, ProTEV,and SUMO protease. Additional exemplary cleaving agents include, but arenot limited to, palladium, clostripain, thrombin, chymotrypsin, trypsin,trypsin-like proteases, carboxypeptidase, enteropeptidase, Kex 2protease, Omp T protease, subtilisin, V8 protease, HIV protease,rhinovirus protease, furilisin protease, IgA proteases, human Paceprotease, collagenase, Nia protease, poliovirus 2Apro protease,poliovirus 3C protease, genenase, furin, elastase, Proteinase K, pepsin,rennin (chymosin), microbial aspartic proteases, papain, calpain,chymopapain, ficin (ficain), bromelain (bromelase), cathespisin B,caspases, thermolysin, Endoprotease Arg-C, Endoprotease Glu-C,Endoprotease Lys-C, kallikrein, and plasmin.

In certain embodiments, the fusion polypeptide is expressed as a solubleprotein or as an inclusion body. In a related embodiment, the disclosurecontemplates isolating the expressed fusion polypeptide from the hostcell or culture medium. In a further embodiment, the isolated fusionpolypeptide is contacted with a cleaving agent as described herein.

In one embodiment, the disclosure provides a bacterial host cellcomprising an expression vector, said vector comprising a polynucleotideencoding a CNP variant peptide linked to a polynucleotide encoding acleavable peptide or protein. In some embodiments, the cleavable peptideor protein is selected from the group consisting of a histidine tag,human transcription factor TAF12, ketosteroid isomerase, maltose-bindingprotein, β-galactosidase, glutathione-S-transferase, thioredoxin, chitinbinding domain, and BMP-2 mutation, or fragments thereof.

In another embodiment, the host cell is a bacteria, such as E. coli. Ina related embodiment, the E. coli cell is selected from the groupconsisting of BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pGro7,ArcticExpress(DE3), C41 [also called C41(DE3)], C43 [also calledC43(DE3)], Origami B(DE3), Origami B(DE3)pLysS, KRX, and Tuner(DE3). Instill a further embodiment, the host cell comprises a vector asdescribed above. In some embodiments, the host cell is transformed withthe vector prior to cell culture.

In certain embodiments, it is contemplated that the host cell iscultured in a medium under conditions suitable for expression of afusion polypeptide encoded by the polynucleotides. In one embodiment,the fusion polypeptide is expressed as a soluble protein or as inclusionbody. In a related embodiment, the expressed fusion polypeptide isisolated from the host cell or culture medium. In still anotherembodiment, the isolated fusion polypeptide is contacted with a cleavingagent as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows expression of CNP fusion proteins in E. coli (FIG. 1A:Coomassie blue stain, FIG. 1B: Western blot). M: protein marker; T:total cell lysates; S: soluble supernatants; P: 2 ug CNP22; KSI:KSI-CNP(M/N) fusion protein expression (insoluble); N: Un-inducedKSI-CNP fusion protein total lysates; KSI′: KSI-P-CNP fusion proteinexpression (insoluble); Trx: Trx-P-CNP fusion protein expression(soluble); MBP: MBP-P-CNP fusion protein expression (soluble); TAF:TAF-P-CNP fusion protein expression (insoluble) (BL21); TAF′: TAF-P-CNPfusion protein expression (insoluble) BL21(DE3), where CNP is Gly-CNP37.

FIG. 2 shows formic acid cleavage of TAF-CNP inclusion bodies. M:protein marker; 1: Gly-CNP37 positive control; 2: uncleaved TAF-CNPinclusion bodies; 3: 2% formic acid cleaved TAF-CNP inclusion bodies,where CNP is Gly-CNP37.

FIGS. 3A-E depict expression of CNP fusion proteins in E. coli. M:protein marker; Tu: total un-induced cell lysates; Su: un-inducedsoluble supernatants; T: total induced cell lysates; S: solublesupernatants; C1: CNP22; C: Gly-wtCNP37 (“CNP38”); P: insoluble pellets.A: KSI: KSI-CNP38(M/N) fusion protein expression (Insoluble); KSI′:KSI-Pro-CNP38 (Pro-Gly-wtCNP37 is designated “Pro-CNP38”) fusion proteinexpression (Insoluble); Trx: Trx-Pro-CNP38 fusion expression (Soluble);MBP: MBP-Pro-CNP38 fusion protein expression (Soluble); TAF:TAF-Pro-CNP38 fusion protein expression (Insoluble) from BL21 cell;TAF′: TAF-Pro-CNP38 fusion protein expression (Insoluble) from BL21(DE3)cell. B: TAF-Pro-CNP37 and BMP-Pro-CNP37 fusion protein expression. C:BMP-Pro-CNP38 fusion protein and BMP protein expression. D:TAF-Pro-HSA-CNP (“Pro-GHKSEVAHRFK-wtCNP27 (SEQ ID NO: 188) is designated“Pro-HSA-CNP”) fusion protein expression. E: TAF-Pro-CNP38 fusionprotein and TAF protein expression.

FIGS. 4A-C depict formic acid cleavage of TAF-Pro-CNP38 inclusionbodies. A: 50% formic acid cleavage of TAF-Pro-CNP38 inclusion bodies.M: protein marker; U: un-cleaved TAF-Pro-CNP38 inclusion bodies; 25° C.,37° C., 42° C., 55° C.: TAF-Pro-CNP38 inclusion bodies were cleaved in50% formic acid at 25° C., 37° C., 42° C. or 55° C. for 24 hours. 37°C.-S and 55° C.-S: soluble supernatants from the cleavage reactions at37° C. and 55° C. neutralized with 10N NaOH and centrifuged at 14,000rpm for 15 minutes. B: 10% and 2% formic acid cleavage of TAF-Pro-CNP38inclusion bodies. M: protein marker; U: un-cleaved TAF-Pro-CNP38inclusion bodies; C: formic acid cleaved TAF-Pro-CNP38; S: solublesupernatant after centrifuged at 14,000 rpm for 5 minutes withoutneutralization; P: insoluble pellet after centrifuged at 14,000 rpm for5 minutes without neutralization. C: LC/MS analysis of 2% and 10% formicacid cleaved products from TAF-Pro-CNP38 inclusion bodies.

FIGS. 5A-C depict formic acid cleavage of TAF-Pro-CNP38 inclusion bodiesat different temperature and time of formic acid cleavage. M: proteinmarker; C: Gly-wtCNP37 (“CNP38”) positive control; U: un-cleavedTAF-Pro-CNP38 inclusion bodies. A: 2% formic acid cleaved TAF-Pro-CNP38at 42° C., 55° C. or 70° C. for 6, 24 or 48 hours. B: 2% formic acidcleaved TAF-Pro-CNP38 at 55° C., 60° C., 65° C. or 70° C. for 17 or 24hours. C: LC/MS analysis of 2% formic acid cleaved products fromTAF-Pro-CNP38 inclusion bodies.

FIG. 6 is an SDS-PAGE (Coomassie blue stain) for the expression of aTAF-CNP34 fusion protein. M: protein marker; C: control [Gly-CNP37(“CNP38”)]; T: total cell lysates; S: supernatant; TI: total celllysates induced; SI: supernatant induced.

FIG. 7 is an SDS-PAGE (Coomassie blue stain) for the expression offusion proteins TAF-NL-(C/A & 6D/6E)-Pro-CNP38, TAF(C/A &10D/10E)-Pro-CNP38, and TAF-Pro-CNP53, where “Pro-CNP38” denotesPro-Gly-CNP37. M: protein marker; C: control [Gly-CNP37 (“CNP38”)]; T:total cell lysates; TI: total cell lysates induced; S: supernatant; SI:supernatant induced.

FIG. 8 is an SDS-PAGE (Coomassie blue stain) of the products of formicacid cleavage of the fusion proteins TAF-CNP34 and TAF-Pro-CNP53. M:protein marker; P: positive control [Gly-CNP37 (“CNP38”)]; U: uncleaved;C: cleaved; CS: cleaved supernatant; CP: cleaved pellet.

FIG. 9 is an LC/MS chromatogram showing the peak for CNP-34 after formicacid cleavage of TAF-CNP34.

FIG. 10 is an LC/MS chromatogram showing the peak for Pro-CNP53 afterformic acid cleavage of TAF-Pro-CNP53.

FIG. 11 is an SDS-PAGE (Coomassie blue stain) for the expression offusion proteins TAF(C/A & 4D/4E)-Pro-CNP38 and TAF(4D/4E)-Pro-CNP38,where “Pro-CNP38” denotes Pro-Gly-CNP37. M: protein marker; C: control[Gly-CNP37 (“CNP38”)]; T: total cell lysates; TI: total cell lysatesinduced; S: supernatant; SI: supernatant induced.

FIG. 12 is an SDS-PAGE (Coomassie blue stain) of the products of formicacid cleavage of the fusion proteins TAF(4D/4E)-Pro-CNP38 and TAF(C/A &4D/4E)-Pro-CNP38, where “Pro-CNP38” denotes Pro-Gly-CNP37. M: proteinmarker; P: positive control [Gly-CNP37 (“CNP38”)]; U: uncleaved; C:cleaved; CS: cleaved supernatant; CP: cleaved pellet.

FIG. 13 is an SDS-PAGE (Coomassie blue stain) of the products of formicacid cleavage of the fusion proteins TAF-NL-(C/A & 6D/6E)-Pro-CNP38 andTAF(C/A & 10D/10E)-Pro-CNP38, where “Pro-CNP38” denotes Pro-Gly-CNP37.M: protein marker; P: positive control [Gly-CNP37 (“CNP38”)]; U:uncleaved; C: cleaved; CS: cleaved supernatant; CP: cleaved pellet.

FIG. 14 is a Western blot, using an anti-CNP antibody, of aTAF-Pro-CNP38 fusion protein produced in a 10 L fermentation ofBL21(DE3) cells, where the cells were induced at OD₆₀₀=64 and hour 17and “Pro-CNP38” denotes Pro-Gly-CNP37.

FIG. 15 is an SDS-PAGE (Coomassie blue stain) of eluate fractions fromSP-Sepharose cation-exchange column chromatography of a cruderPro-Gly-CNP37 (“Pro-CNP38”) product. A: TAF-Pro-CNP38 inclusion body(IB) in water; B: IB in formate; C: IB in formate, neutralized; D:neutralized pellet; E: neutralized supernatant; F: TMAE Hi-CAP load; G:TMAE Hi-CAP flow through/SP-Sepharose load; H: SP-Sepharose flowthrough; SP-Sepharose eluate fractions 1-47: 10 uL/lane.

FIG. 16 shows the degree of resistance of N-terminal PEGylated CNP22conjugates to neutral endopeptidase (NEP) in vitro.

FIG. 17 depicts the degree of NEP resistance of CNP variants having anamino acid extension at the N-terminus [“CNP27” is GANRR-CNP22(K4R) (SEQID NO: 36)].

FIG. 18 illustrates the degree of NEP resistance of N-terminal PEGylatedCNP17 and GANRR-CNP22(K4R) (“CNP27”) (SEQ ID NO: 36).

FIG. 19 illustrates the degree of NEP resistance of wtCNP22 and CNPvariants Gly-CNP37, GHKSEVAHRFK-wtCNP27 (“CNP27-HSA” in the figures)(SEQ ID NO: 144) and PEO12-GANRR-CNP22(K4R) (“CNP27-PEO12” in thefigures) (SEQ ID NO: 36).

FIG. 20 shows the ability of CNP variants having an N-terminal aminoacid extension to stimulate cGMP production in NIH3T3 cells in vitro.The results are relative to the level of cGMP produced in the presenceof 1 uM CNP22. “CNP27” is GANRR-CNP22(K4R) (SEQ ID NO: 36)

FIG. 21 displays the ability of N-terminal PEGylated CNP17 andGANRR-CNP22(K4R) (“CNP27”) (SEQ ID NO: 36) to stimulate cGMP productionin NIH3T3 cells.

FIG. 22 illustrates the effects of N-terminal PEGylation of CNP22 oncGMP production.

FIG. 23 illustrates the cGMP production induced by wtCNP22 and CNPvariants Gly-CNP37, GHKSEVAHRFK-wtCNP27 (“CNP27-HSA”) (SEQ ID NO: 144),wtCNP29 and PEO12-GANRR-CNP22(K4R) (“CNP27-PEO12”) (SEQ ID NO: 36) inNIH3T3 cells.

FIGS. 24A and B show that CNP-22 and Pro-Gly-CNP37 (“Pro-CNP38”)stimulated cGMP production through NPR-B with similar dose-responsecurves, and to a much greater extent than through NPR-A, and exhibited asimilar profile for NPR-B vs. NPR-C selectivity in in vitro signalingcompetition assays.

FIG. 25 demonstrates that exposure of rat chondrosarcoma cells to CNP221 hour once daily or 2 hours once daily has substantially similareffectiveness in reversing FGF2-induced arrest of chondrocyte growth ascontinuous exposure to CNP22.

FIGS. 26A and B show results from a dose response study of CNP22 effectson FGF2-arrested rat chondrosarcoma (RCS) cells.

FIGS. 27A-D show that addition of CNP22 to RCS cells arrested by FGF2increases matrix synthesis and partly inhibits FGF2, as assessed by³⁵S-sulfate and ³H-Pro incorporation into or decrease from matrix.Panels A and C, synthesis; B and D, degradation; A and B, ³⁵Smeasurement; C and D, ³H measurement. Statistically significantdifferences are highlighted (ANOVA; *p<0.05, **p<0.01).

FIGS. 28A-C show the levels of aggrecan and fibronectin production(mRNA, panels A and C, and protein, panel B) in RCS cells cultured withFGF2 and CNP22.

FIG. 29 shows the efficacy of CNP37 and PEO24-GANRR-CNP22(K4R)(“CNP27-PEO24” in the figures) (SEQ ID NO: 36) in stimulatinglongitudinal growth of wild-type femur in an ex vivo mouse organ model.

FIG. 30 shows longitudinal bone growth of 2-3 day-old wild-type mousetibias treated with CNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (SEQ ID NO:36) every two days. Results are normalized to measurements prior totreatment (day 0). Data is represented as means±SEM (n=8).

FIG. 31 shows longitudinal bone growth of 2-3 day old achondroplasticFGFR3^(ach) mouse tibias treated with CNP22, CNP37 orPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) every two days. Results arenormalized to measurements prior to treatment (day 0). Data isrepresented as means±SEM (n=7-8).

FIG. 32 shows longitudinal bone growth of 2-3 day-old wild-type mousefemurs treated with CNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (SEQ ID NO:36) every two days. Results are normalized to measurements prior totreatment (day 0). Data is represented as means±SEM (n=8).

FIG. 33 shows longitudinal bone growth of 2-3 day-old FGFR3^(ach) mousefemurs treated with CNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (SEQ ID NO:36) every two days. Results are normalized to measurements prior totreatment (day 0). Data is represented as means±SEM (n=3-7).

FIGS. 34A-I depict CNP37 biodistribution in FGFR3^(ach) mouse femurstreated ex vivo every two days. Panels A-C illustrate distribution indistal femurs, panels D-F illustrate distribution in articularchondrocytes and panels G-I illustrate distribution in hypertrophicchondrocytes.

FIGS. 35A-C depict the cellularity of proliferating columns in growthplates after treatment of wild-type and FGFR3^(ach) mouse femurs withCNP22 or CNP37 every two days for 8 days. (A) no treatment, (B) cellnumbers per column after treatment, (C) morphological studies aftertreatment. The panels in FIGS. 35C(i) to C(vi). correspond to the sampleorder set out in FIG. 35B. Data is represented as means±SEM (n=4-8).

FIGS. 36A-C depict chondrocyte hypertrophy after ex vivo treatment ofwild-type and FGFR3^(ach) mouse femurs with CNP22 or CNP37 every twodays for 8 days. (A) no treatment, (B) cell size after treatment, (C)morphological studies after treatment. The panels in FIGS. 36C(i) toC(vi) correspond to the sample order set out in FIG. 36B. Data isrepresented as means±SEM (n=4-9).

FIGS. 37A-I depict the biodistribution of CNP37 in FGFR3^(ach) mousetibias treated in vivo. Panels A-C show distribution in distal femurs,panels D-F illustrate distribution in articular chondrocytes and panelsG-I illustrate distribution in hypertrophic chondrocytes.

FIGS. 38A-C illustrate the in vivo effects of CNP37 on FGFR3^(ach) mousetibia growth plate: (A) total growth plate thickness, (B) proliferatingzone thickness, and (C) hypertrophic zone thickness. Data is representedas means±SEM (n=7-15).

FIG. 39 shows cGMP levels in conditioned media from wild-type mousefemurs treated ex vivo with CNP22, CNP37 or PEO24-GANRR-CNP22(K4R)(“CNP27-PEO24”) (SEQ ID NO: 36) (p<0.01).

FIG. 40 shows cGMP levels in conditioned media from FGFR3^(ach) mousefemurs treated ex vivo with CNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (SEQID NO: 36) (p<0.01).

FIG. 41 depicts cGMP levels in conditioned media from wild-type mousetibias treated ex vivo with CNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (SEQID NO: 36) (p<0.01).

FIG. 42 shows cGMP levels in conditioned media from FGFR3^(ach) mousetibias treated ex vivo with CNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (SEQID NO: 36) (p<0.01).

FIG. 43 demonstrates that ex vivo exposure of wild-type and FGFR3^(ach)mouse femurs to CNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36)increased the levels of cleaved collagen type II in the conditionedmedia (p<0.05).

FIG. 44 depicts the hypertrophic region of femoral bones isolated fromwild-type mice and FGFR3^(Y367C) mice (a mouse model of severeachondroplasia) and treated ex vivo with vehicle or 1 uM Pro-Gly-CNP37(“ProCNP38”) for 6 days, demonstrating that Pro-Gly-CNP37 treatmentresulted in increase in bone growth and expansion in the growth plate.

FIG. 45 shows that CNP37 and PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36)intravenously (i.v.) administered to rats have a much longer half-lifeand a much greater bioavailability in the plasma than CNP22.

FIG. 46 illustrates that PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36)subcutaneously (s.c.) administered to rats also has a much longerhalf-life and a much greater bioavailability in the plasma than CNP22.

FIG. 47 demonstrates that i.v. administered CNP37 andPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) stimulate a much greater level ofcGMP production in rats than CNP22.

FIG. 48 shows that s.c. administered PEO24-GANRR-CNP22(K4R) (SEQ ID NO:36) and, to a lesser extent, CNP37 are substantially more effective instimulating cGMP production in rats than CNP22.

FIG. 49 shows body weight measurements of wild-type mice treated withGly-CNP37 or PEO12-GANRR-CNP22(K4R) (SEQ ID NO: 36).

FIG. 50 shows tail length measurements of wild-type mice treated withGly-CNP37 or PEO12-GANRR-CNP22(K4R) (SEQ ID NO: 36).

FIG. 51 illustrates the effect of treatment of FGFR3^(ach) mice withCNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) on body length(p=0.02, 1-tailed t-test, unequal variance).

FIG. 52 shows the effect of CNP22, CNP37 and PEO24-GANRR-CNP22(K4R) (SEQID NO: 36) on tail length in FGFR3^(ach) mice.

FIGS. 53A and B show the effect of CNP22, CNP37 andPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) on the length of distal longbones (A, ulna; B, tibia) in FGFR3^(ach) mice (p<0.01, one-tailedt-test, unequal variance).

FIGS. 54A and B show the effect of CNP22, CNP37 andPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) on the length of proximal bones(A, humerus; B, femur) in FGFR3^(ach) mice (p<0.01, one-tailed t-test,unequal variance).

FIG. 55 illustrates that CNP37 administration corrects rhizomelia(disproportion of the length of the proximal limbs) as assessed by thefemur:tibia ratio observed in FGFR3^(ach) mice (p<0.01, one-tailedt-test, unequal variance).

FIG. 56 shows the effect of CNP22, CNP37 and PEO24-GANRR-CNP22(K4R) (SEQID NO: 36) on head length in FGFR3^(ach) mice (p<0.01 one-tailed t-test,unequal variance).

FIG. 57 shows that treatment of FGFR3^(ach) mice with CNP37 increasesthe size of the external auditory meatus (EAM) (P=0.03, one-tailedt-test, unequal variance).

FIG. 58 shows the effect of CNP22, CNP37 and PEO24-GANRR-CNP22(K4R) (SEQID NO: 36) on spinal length in achondroplasic mice, expressed asextension of vertebral bodies (e.g., lumbar vertebra 5).

FIG. 59 shows that treatment of FGFR3^(ach) mice with CNP37 orPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) results in increased cGMP plasmalevels 15-min post-dose.

FIG. 60 illustrates the serum levels of cleaved collagen type II inFGFR3^(ach) mice treated 5 weeks with CNP22, CNP37 orPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36).

FIG. 61 shows the serum levels of osteocalcin in FGFR3^(ach) micetreated 5 weeks with CNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (SEQ ID NO:36).

FIG. 62 shows cGMP plasma levels 15-min post-dose from wild-type micetreated with Gly-CNP37 or PEO12-GANRR-CNP22(K4R) (SEQ ID NO: 36)(p<0.05).

FIG. 63 shows the serum levels of cleaved collagen type II in wild-typemice treated 5 weeks with Gly-CNP37 or PEO12-GANRR-CNP22(K4R) (SEQ IDNO: 36).

FIGS. 64-66 display the levels of cGMP, cleaved collagen type II andalkaline phosphatase after administration of vehicle or 20 nmol/kg or 70nmol/kg Pro-Gly-CNP37 (“Pro-CNP38”) to wild-type mice.

FIGS. 67 and 68 depict cleaved collagen type II and total alkalinephosphatase levels after administration of vehicle or Pro-Gly-CNP37(“Pro-CNP38”) under different dosing regimens.

FIG. 69 illustrates the relative increase in body length ofPro-Gly-CNP37 (“Pro-CNP38”) and vehicle treated animals in two separatestudies (S1 and S2) at Day 37 vs Day 1.

FIGS. 70A and B show changes in bone mineral density (A) and bonemineral content (B) after administration of Pro-Gly-CNP37 (“Pro-CNP38”).

FIG. 71 shows plasma cGMP levels 15 min after the last subcutaneous doseof CNP variant, Day 36, where Gly-wtCNP37 is “CNP38”, Pro-Gly-wtCNP37 is“Pro-CNP38”, and GHKSEVAHRFK-wtCNP27 (SEQ ID NO: 144) is “HSA-CNP27” inFIGS. 71-73.

FIG. 72 shows serum levels of cleaved collagen type II from mice treatedwith Gly-CNP37, Pro-Gly-CNP37 or GHKSEVAHRFK-CNP27 (SEQ ID NO: 144).

FIG. 73 shows serum levels of alkaline phosphatase from mice treatedwith Gly-CNP37, Pro-Gly-CNP37 or GHKSEVAHRFK-CNP27 (SEQ ID NO: 144).

FIGS. 74A and B depict desensitization of the cGMP response after acute(A) or chronic (B) treatment with 1 uM Gly-CNP37.

FIG. 75A demonstrates that daily treatment of wild-type mice with 200nmol/kg of Pro-Gly-CNP37 (“Pro-CNP38”) for 8 days did not desensitizethe cGMP response. FIG. 75B shows that treatment of the mice with 200nmol/kg of Pro-Gly-CNP37 once a day for two consecutive days potentiatedthe cGMP response.

FIGS. 76A-D show that treatment of wild-type mice with 200 nmol/kg ofGly-CNP37 stimulated cGMP secretion in distal femurs (cartilage andbone) (A), femoral cortices (bone) (B), ear pinna (cartilage) (C), andkidney (D). FIGS. 76E-H show that liver (E), heart (F), lung (G) andbrain (H) tissues did not exhibit appreciable cGMP secretion in responseto Gly-CNP37 relative to vehicle control at the studied time points.

FIGS. 77-82 show results from an on-going study in normal juvenilecynomolgus monkeys subcutaneously injected daily with vehicle or 10 or36 ug/kg of Pro-Gly-CNP37. Both doses of Pro-Gly-CNP37 have increasedgrowth plate width (FIG. 77), increased right and left tibia lengths(FIGS. 78A and B), increased leg length (FIG. 79), increased arm length(FIG. 80), increased body length (FIG. 81), and increased the serumlevel of alkaline phosphatase (FIG. 82).

FIG. 83 depicts the observed plot of degradation rate constant (K_(obs))vs. pH at pH 3-8 and 5° C., 25° C. and 40° C. for Gly-CNP37formulations.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to novel variants of CNP having reducedaffinity to NEP and/or NPR-C, and reduced susceptibility to cleavage byNEP and/or clearance by NPR-C, pharmaceutical compositions comprisingsuch CNP variants, and methods of using such

CNP variants to treat disorders responsive to CNP, including but notlimited to bone-related disorders such as achondroplasia and disordersassociated with vascular smooth muscle cells and tissues.

A. Definitions

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow.

As used in the specification and the appended claims, the indefinitearticles “a” and “an” and the definite article “the” include plural aswell as singular referents unless the context clearly dictatesotherwise.

The term “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined. Incertain embodiments, the term “about” or “approximately” means within 1,2, 3, or 4 standard deviations. In certain embodiments, the term “about”or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Wheneverthe term “about” or “approximately” precedes the first numerical valuein a series of two or more numerical values, it is understood that theterm “about” or “approximately” applies to each one of the numericalvalues in that series.

The terms “ambient temperature” and “room temperature” are usedinterchangeably herein and refer to the temperature of the surroundingenvironment (e.g., the room in which a reaction is conducted or acomposition is stored). In certain embodiments, ambient temperature orroom temperature is a range from about 15° C. to about 28° C., or fromabout 15° C. to about 25° C., or from about 20° C. to about 28° C., orfrom about 20° C. to about 25° C., or from about 22° C. to about 28° C.,or from about 22° C. to about 25° C. In other embodiments, ambienttemperature or room temperature is about 15° C., 16° C., 17° C., 18° C.,19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C.or 28° C.

Definition of standard chemistry terms may be found in reference works,including Carey and Sundberg, Advanced Organic Chemistry, 3^(rd)Edition, Vols. A and B (Plenum Press, New York 1992). The practice ofthe present disclosure may employ, unless otherwise indicated, certainconventional methods of synthetic organic chemistry, mass spectrometry,preparative and analytical chromatography, protein chemistry,biochemistry, recombinant DNA technology and pharmacology, within theskill of the art. See, e.g., T. E. Creighton, Proteins: Structures andMolecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger,Biochemistry (Worth Publishers, Inc., 4^(th) Edition, 2004); Sambrook,et al., Molecular Cloning: A Laboratory Manual (2^(nd) Edition, 1989);Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press,Inc.); Remington's Pharmaceutical Sciences, 18^(th) Edition (Easton,Pa.: Mack Publishing Company, 1990).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

The following amino acid abbreviations are used throughout the text:

Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid:Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E)Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L)Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro(P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr(Y) Valine: Val (V)

“Polypeptide” and “protein” refer to a polymer composed of amino acidresidues, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof, linked via peptide bonds orpeptide bond isosteres. Synthetic polypeptides can be synthesized, forexample, using an automated polypeptide synthesizer. The terms“polypeptide” and “protein” are not limited to a minimum length of theproduct. The term “protein” typically refers to large polypeptides. Theterm “peptide” typically refers to short polypeptides. Thus, peptides,oligopeptides, dimers, multimers, and the like, are included within thedefinition. Both full-length proteins and fragments thereof areencompassed by the definition. The terms “polypeptide” and “protein”also include postexpression modifications of the polypeptide or protein,for example, glycosylation, acetylation, phosphorylation and the like.Furthermore, for purposes of the present disclosure, a “polypeptide” caninclude “modifications,” such as deletions, additions, substitutions(which may be conservative in nature or may include substitutions withany of the 20 amino acids that are commonly present in human proteins,or any other naturally or non-naturally-occurring or atypical aminoacids), and chemical modifications (e.g., addition of or substitutionwith peptidomimetics), to the native sequence. These modifications maybe deliberate, as through site-directed mutagenesis, or through chemicalmodification of amino acids to remove or attach chemical moieties, ormay be accidental, such as through mutations arising with hosts thatproduce the proteins or through errors due to PCR amplification.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

“Conservative substitution” refers to substitution of an amino acid in apolypeptide with a functionally, structurally or chemically similarnatural or unnatural amino acid. In one embodiment, the following groupseach contain natural amino acids that are conservative substitutions forone another:

(1), Alanine (A) Serine (S), Threonine (T);

(2) Aspartic acid (D), Glutamic acid (E);

(3) Asparagine (N), Glutamine (Q);

(4) Arginine (R), Lysine (K);

(5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

(6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

In another embodiment, the following groups each contain natural aminoacids that are conservative substitutions for one another:

(1) Glycine (G), Alanine (A);

(2) Aspartic acid (D), Glutamic acid (E);

(3) Asparagine (N), Glutamine (Q);

(4) Arginine (R), Lysine (K);

(5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), Alanine(A);

(6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); and

(7) Serine (S), Threonine (T), Cysteine (C).

In a further embodiment, amino acids may be grouped as set out below.

(1) hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence backbone orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe, His.

In one embodiment, the peptides or polypeptides described herein aregenerated via recombinant means, using a polynucleotide encoding a CNPvariant. The disclosure thus encompasses polynucleotides encoding any ofthe CNP variants described herein, host cells or vectors comprising suchpolynucleotides, optionally linked to expression control sequences, andmethods of using such polynucleotides, vectors or host cells to produceCNP variants of the disclosure. CNP variants expressed by suchpolynucleotides may be produced by methods including growing host cellsin culture medium under conditions suitable for expression of thepolynucleotide encoding a CNP variant, and isolating the expressionproduct from the host cells or culture medium. Actual expressionproducts may vary slightly from the encoded protein product depending onany post-translational processing.

“Polynucleotide” refers to a polymer composed of nucleotide units.Polynucleotides include naturally occurring nucleic acids, such asdeoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well asnucleic acid analogs. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), the nucleotide sequence also encompasses anRNA sequence (i.e., A, U, G, C) in which “U” replaces “T.” “cDNA” refersto a DNA that is complementary or identical to an mRNA, in either singlestranded or double stranded form.

“Expression control sequence” refers to a nucleotide sequence thatregulates the expression of a nucleotide sequence operatively linkedthereto. “Operatively linked” refers to a functional relationshipbetween two parts in which the activity of one part (e.g., the abilityto regulate transcription) results in an action on the other part (e.g.,transcription of the sequence). Expression control sequences caninclude, for example and without limitation, sequences of promoters(e.g., inducible or constitutive), enhancers, transcription terminators,a start codon (i.e., ATG), splicing signals for introns, and stopcodons.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell. A host cell thatcomprises the recombinant polynucleotide is referred to as a“recombinant host cell.” The gene is then expressed in the recombinanthost cell to produce, e.g., a “recombinant polypeptide.” A recombinantpolynucleotide may serve a non-coding function (e.g., promoter, originof replication, ribosome-binding site, etc.) as well.

“Chimera” as used herein refers to a polynucleotide or polypeptidecomprising at least two heterologous polynucleotide or polypeptidesequences (i.e. derived from different sources or not associated witheach other as a naturally-occurring sequence) which are directly orindirectly attached or linked together using techniques commonly knownin the art, e.g., recombinant expression or chemical crosslinking. Inone embodiment, the heterologous sequence can comprise a protein orpeptide directly or indirectly linked to a CNP peptide or variant,including proteins or peptides which are cleavable from the CNP peptideor variant. In a related embodiment, CNP variants are chimera asdescribed herein.

In certain embodiments, chimeras include CNP fusion proteins comprisinga cleavable carrier protein or peptide tag. The term “cleavable carrierprotein” or “cleavable peptide tag” refers to a peptide or polypeptidesequence that may be fused, directly or indirectly via a linker, to aheterologous polypeptide sequence, and is removable from theheterologous sequence using an agent that cleaves or separates thecleavable peptide or polypeptide from the heterologous polypeptide orprotein. In some embodiments, the cleavable carrier protein or peptidetag improves generation, purification and/or detection of the fusionprotein or the heterologous polypeptide. Exemplary cleavable carrierproteins and peptide tags include, but are not limited to, humantranscription factor TAF12 (TAF12), ketosteroid isomerase (KSI),maltose-binding protein (MBP), β-galactosidase (β-Gal),glutathione-S-transferase (GST), thioredoxin (Trx), chitin-bindingdomain (CBD), BMP-2 mutation (BMPM), SUMO, CAT, TrpE, staphylococcalprotein A, streptococcal proteins, starch-binding protein,cellulose-binding domain of endoglucanase A, cellulose-binding domain ofexoglucanase Cex, biotin-binding domain, recA, Flag, c-Myc, poly(His),poly(Arg), poly(Asp), poly(Gln), poly(Phe), poly(Cys), green fluorescentprotein, red fluorescent protein, yellow fluorescent protein, cyanfluorescent protein, biotin, avidin, streptavidin, antibody epitopes,and fragments thereof.

A “cleaving agent” is an agent that is useful to cleave or separate,e.g., a cleavable peptide or polypeptide from a heterologous polypeptideor protein. Exemplary cleaving agents include, but are not limited to,palladium, cyanogen bromide (CNBr), formic acid, hydroxylamine,clostripain, thrombin, chymotrypsin, trypsin, trypsin-like proteases,carboxypeptidase, enterokinase (enteropeptidase), Kex 2 protease, Omp Tprotease, Factor Xa protease, subtilisin, proTEV, SUMO protease, V8protease, HIV protease, rhinovirus protease, furilisin protease, IgAproteases, human Pace protease, collagenase, Nia protease, poliovirus2Apro protease, poliovirus 3C protease, genenase, furin, elastase,Proteinase K, pepsin, rennin (chymosin), microbial aspartic proteases,papain, calpain, chymopapain, ficin (ficain), bromelain (bromelase),cathespisin B, caspases, thermolysin, Endoprotease Arg-C, EndoproteaseGlu-C, Endoprotease Lys-C, kallikrein, and plasmin.

The terms “identical” and percent “identity”, in the context of two ormore polynucleotide or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially homologous” or “substantially identical”, inthe context of two nucleic acids or polypeptides, generally refers totwo or more sequences or subsequences that have at least 40%, 50%, 60%,70%, 80%, 90%, 95%, or 98% nucleotide or amino acid residue identity,when compared and aligned for maximum correspondence, as measured usingone of the following sequence comparison algorithms or by visualinspection. In certain embodiments, the substantial homology or identityexists over regions of the sequences that are at least about 25, 50, 100or 150 residues in length. In another embodiment, the sequences aresubstantially homologous or identical over the entire length of eitheror both comparison biopolymers.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are inputted into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math., 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch,J. Mol. Biol., 48: 443 (1970), by the search for similarity method ofPearson & Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection. Oneexample of a useful algorithm is PILEUP, which uses a simplification ofthe progressive alignment method of Feng & Doolittle, J. Mol. Evol., 35:351-360 (1987) and is similar to the method described by Higgins &Sharp, CABIOS, 5: 151-153 (1989). Another algorithm useful forgenerating multiple alignments of sequences is Clustal W (Thompson etal., Nucleic Acids Research, 22: 4673-4680 (1994)). An example of analgorithm that is suitable for determining percent sequence identity andsequence similarity is the BLAST algorithm (Altschul et al., J. Mol.Biol., 215: 403-410 (1990); Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA, 89: 10915 (1989); Karlin & Altschul, Proc. Natl. Acad. Sci. USA,90: 5873-5787 (1993)). Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation.

A further indication that two nucleic acid sequences or polypeptides aresubstantially homologous or identical is that the polypeptide encoded bythe first nucleic acid is immunologically cross reactive with thepolypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two polypeptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described herein.

“Substantially pure” or “isolated” means an object species is thepredominant species present (i.e., on a molar basis, more abundant thanany other individual macromolecular species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50% (on a molar basis) of allmacromolecular species present. In one embodiment, a substantially purecomposition means that the species of interest comprises at least about70%, 75%, 80%, 85%, 90%, 95%, 98% or more of the macromolecular speciespresent in the composition on a molar or weight basis. The objectspecies is purified to essential homogeneity (contaminant species cannotbe detected in the composition by conventional detection methods) if thecomposition consists essentially of a single macromolecular species.Solvent species, small molecules (<500 Daltons), stabilizers (e.g.,BSA), and elemental ion species are not considered macromolecularspecies for purposes of this definition. In an embodiment, the compoundsof the disclosure are substantially pure or isolated. In anotherembodiment, the compounds of the disclosure are substantially pure orisolated with respect to the macromolecular starting materials used intheir production. In yet another embodiment, the pharmaceuticalcompositions of the disclosure comprise a substantially pure or isolatedCNP variant admixed with one or more pharmaceutically acceptableexcipients, carriers or diluents, and optionally with anotherbiologically active agent.

“Naturally occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) and which has not been intentionally modified by man in thelaboratory is naturally occurring. In one embodiment, a “naturallyoccurring” substance is of human origin.

“Wild-type” (wt) is a term referring to the natural form, includingsequence, of a polynucleotide, polypeptide or protein in a species. Awild-type form is distinguished from a mutant form of a polynucleotide,polypeptide or protein arising from genetic mutation(s).

In one embodiment, a first polypeptide that is an “analog” or “variant”or “derivative” of a second polypeptide is a polypeptide having at leastabout 50%, 60% or 70% sequence homology, but less than 100% sequencehomology, with the second polypeptide. Such analogs, variants orderivatives may be comprised of non-naturally occurring amino acidresidues, including without limitation, homoarginine, ornithine,penicillamine, and norvaline, as well as naturally occurring amino acidresidues. Such analogs, variants or derivatives may also be composed ofone or a plurality of D-amino acid residues, and may also containpeptidomimetics or peptide bond isosteres such as non-peptide linkagesbetween two or more amino acid or peptidomimetic residues. In anotherembodiment, a first polypeptide is an “analog”, “variant” or“derivative” of a second polypeptide if the first polypeptide is not aknown cleavage product of the second polypeptide or is not a knownprecursor of the second polypeptide, even if the first polypeptide has100% sequence homology to the second polypeptide or has a wild-typesequence.

In an embodiment, the term “derived from” as used herein refers to apolypeptide or peptide sequence that is based on a wild type ornaturally occurring polypeptide or peptide sequence and can have one ormore deletions, additions, and/or substitutions with natural aminoacids, unnatural amino acids or peptidomimetics. In one embodiment, thederivative sequence shares at least about 40%, 50%, 60% or 70%, but lessthan 100%, sequence similarity to the wild-type or naturally occurringsequence. In another embodiment, the derivative may be a fragment of apolypeptide, wherein the fragment is substantially homologous (e.g., atleast about 70%, 75%, 80%, 85%, 90%, or 95% homologous) to the wild-typepolypeptide over a length of at least about 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 amino acids. In still another embodiment, a polypeptide is“derived from” a wild-type polypeptide if it has a moiety (e.g., apolymer such as, e.g., PEG) directly or indirectly attached to it whichis not present on the wild-type polypeptide, even if both polypeptidesshare 100% homology in their amino acid sequence.

As used herein, an “NPPC-derived” polypeptide refers to a polypeptidederived from the natriuretic peptide precursor C(NPPC) polypeptide,which is a single chain 126-amino acid pre-pro polypeptide, and whichupon cleavage ultimately results in wtCNP22. Removal of the signalpeptide from NPPC yields pro-CNP, and further cleavage by theendoprotease furin generates an active 53-amino acid peptide (CNP-53),which is secreted and cleaved again by an unknown enzyme to produce themature 22-amino acid peptide (CNP, or CNP-22). Therefore, CNP22 itselfis an “NPPC-derived” polypeptide by virtue of being derived from NPPC.In one embodiment, an NPPC-derived polypeptide is at least about 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% homologous to the wild typeNPPC over the same number of amino acid residues. It is furthercontemplated that an NPPC-derived peptide may comprise from about 1 toabout 53, or 1 to 37, or 1 to 35, or 1 to 31, or 1 to 27, or 1 to 22, orto 35, or about 15 to about 37 residues of the NPPC polypeptide. In oneembodiment, an NPPC-derived peptide may comprise a sequence of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 amino acidsderived from the NPPC polypeptide.

The term “effective amount” means a dosage sufficient to produce adesired result on a health condition, pathology, or disease of a subjector for a diagnostic purpose. The desired result may comprise asubjective or objective improvement in the recipient of the dosage.“Therapeutically effective amount” refers to that amount of an agenteffective to produce the intended beneficial effect on health. Anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation. Itwill be understood that the specific dose level and frequency of dosagefor any particular patient may be varied and will depend upon a varietyof factors, including the activity of the specific compound employed;the bioavailability, metabolic stability, rate of excretion and lengthof action of that compound; the mode and time of administration of thecompound; the age, body weight, general health, sex, and diet of thepatient; and the severity of the particular condition.

“Treatment” refers to prophylactic treatment or therapeutic treatment ordiagnostic treatment. In certain embodiments, “treatment” refers toadministration of a compound or composition to a subject fortherapeutic, prophylactic or diagnostic purposes.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease, for the purpose of decreasing the risk of developing pathology.The compounds or compositions of the disclosure may be given as aprophylactic treatment to reduce the likelihood of developing apathology or to minimize the severity of the pathology, if developed.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs or symptoms of pathology for the purpose of diminishingor eliminating those signs or symptoms. The signs or symptoms may bebiochemical, cellular, histological, functional or physical, subjectiveor objective. The compounds of the disclosure may also be given as atherapeutic treatment or for diagnosis.

“Diagnostic” means identifying the presence, extent and/or nature of apathologic condition. Diagnostic methods differ in their specificity andselectivity. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis.

“Bone- or cartilage-associated biomarker” or “bone- orcartilage-associated marker” refers to a growth factor, enzyme, protein,or other detectable biological substance or moiety whose level isincreased or decreased in association with, e.g., cartilage turnover,cartilage formation, cartilage growth, bone resorption, bone formation,bone growth, or combinations thereof. Such biomarkers may be measuredbefore, during and/or after administration of a CNP variant as describedherein. Exemplary bone- or cartilage-associated biomarkers include, butare not limited to, CNP, cGMP, propeptides of collagen type II andfragments thereof, collagen type II and fragments thereof, propeptidesof collagen type I and fragments thereof, collagen type 1 and fragmentsthereof, osteocalcin, proliferating cell nuclear antigen (PCNA),aggrecan chondroitin sulfate, and alkaline phosphatase. Cartilage- andbone-associated biomarkers can be measured in any appropriate biologicalsample, including but not limited to tissues, blood, serum, plasma,cerebrospinal fluid, synovial fluid and urine. In some embodiments, thebiomarkers are measured in blood, plasma or serum from animalsundergoing efficacy/pharmacodynamic in vivo studies and/or from theconditioned media of ex vivo studies.

In certain embodiments, the level of at least one bone- orcartilage-associated biomarker is measured and the amount or frequencyof administration of CNP variant administered to a subject can beadjusted according to the level of the biomarker measured. In someembodiments, the level of biomarker is “below a target level” or “abovea target level.” A target level of a biomarker is a level or range oflevels of the biomarker at which a therapeutic effect is observed in thesubject receiving the CNP variant. In certain embodiments, the targetlevel of a biomarker for a subject having a CNP-responsive disorder orcondition is the level or range of levels of the biomarker observed in anormal, non-affected subject. In other embodiments, to indicate atherapeutic effect, the target level of a biomarker need not beequivalent to the level or range of levels of the biomarker observed ina normal subject, but can be within, e.g., 100%, 90%, 80%, 70%, 60%,50%, 40%, 30%, 20%, 10% or 5% of the “normal” level or range of levelsof the biomarker observed in a non-affected subject.

For example, if the level of a biomarker increases in association withbone or cartilage formation or growth, the target level of the biomarkerindicating a therapeutic effect may be higher than the level of thebiomarker in patients suffering from a CNP-responsive disorder who havenot been administered a CNP variant, and may optionally be lower thanthe “normal” level(s), at about the “normal” level(s), or above the“normal” level(s) of the biomarker in subjects not suffering from thatdisorder. In one embodiment, if the level of a biomarker is below atarget level, it indicates an inadequate therapeutic effect, which mayrequire an increase in the amount or frequency of administration of CNPvariant administered. In a related embodiment, if the biomarker is abovea target level, it indicates that more CNP variant than necessary hasbeen administered, which may require a decrease in the amount orfrequency of administration of the CNP variant administered.

As another example, if the level of a biomarker decreases in associationwith bone or cartilage formation or growth, the target level of thebiomarker indicating a therapeutic effect may be lower than the level ofthe biomarker in patients suffering from a CNP-responsive disorder whohave not been administered a CNP variant, and may optionally be higherthan the “normal” level(s), at about the “normal” level(s), or below the“normal” level(s) of the biomarker in subjects not suffering from thatdisorder. In such a case, the converse of the above adjustments in CNPvariant amount and frequency of administration may apply.

“Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in subject animal, including humans and mammals. Apharmaceutical composition comprises a therapeutically effective amountof a CNP variant, optionally another biologically active agent, andoptionally a pharmaceutically acceptable excipient, carrier or diluent.In an embodiment, a pharmaceutical composition encompasses a compositioncomprising the active ingredient(s), and the inert ingredient(s) thatmake up the carrier, as well as any product that results, directly orindirectly, from combination, complexation or aggregation of any two ormore of the ingredients, or from dissociation of one or more of theingredients, or from other types of reactions or interactions of one ormore of the ingredients. Accordingly, the pharmaceutical compositions ofthe present disclosure encompass any composition made by admixing acompound of the disclosure and a pharmaceutically acceptable excipient,carrier or diluent.

“Pharmaceutically acceptable carrier” refers to any of the standardpharmaceutical carriers, buffers, and the like, such as a phosphatebuffered saline solution, 5% aqueous solution of dextrose, and emulsions(e.g., an oil/water or water/oil emulsion). Non-limiting examples ofexcipients include adjuvants, binders, fillers, diluents, disintegrants,emulsifying agents, wetting agents, lubricants, glidants, sweeteningagents, flavoring agents, and coloring agents. Suitable pharmaceuticalcarriers, excipients and diluents are described in Remington'sPharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995).Preferred pharmaceutical carriers depend upon the intended mode ofadministration of the active agent. Typical modes of administrationinclude enteral (e.g., oral) or parenteral (e.g., subcutaneous,intramuscular, intravenous or intraperitoneal injection; or topical,transdermal, or transmucosal administration).

A “pharmaceutically acceptable salt” is a salt that can be formulatedinto a compound for pharmaceutical use, including but not limited tometal salts (e.g., sodium, potassium, magnesium, calcium, etc.) andsalts of ammonia or organic amines.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to an individual without causingany undesirable biological effects or without interacting in adeleterious manner with any of the components of the composition inwhich it is contained or with any components present on or in the bodyof the individual.

The term “unit dosage form” refers to physically discrete units suitableas unitary dosages for human and animal subjects, each unit containing apredetermined quantity of a compound of the disclosure calculated in anamount sufficient to produce the desired effect, optionally inassociation with a pharmaceutically acceptable excipient, diluent,carrier or vehicle. The specifications for the novel unit dosage formsof the present disclosure depend on the particular compound employed andthe effect to be achieved, and the pharmacodynamics associated with eachcompound in the host.

“Physiological conditions” refer to conditions in the body of an animal(e.g., a human). Physiological conditions include, but are not limitedto, body temperature and an aqueous environment of physiologic ionicstrength, pH and enzymes. Physiological conditions also encompassconditions in the body of a particular subject which differ from the“normal” conditions present in the majority of subjects, e.g., whichdiffer from the normal human body temperature of approximately 37° C. ordiffer from the normal human blood pH of approximately 7.4.

By “physiological pH” or a “pH in a physiological range” is meant a pHin the range of approximately 7.0 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, the term “subject” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, any member of themammalian class: humans, non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish, and the like. The term does not denote a particular ageor gender.

The terms “polyethylene glycol”, “PEG”, “polyethylene oxide” and “PEO”are used interchangeably herein unless indicated otherwise. A CNPpeptide (CNP22 or a variant thereof) conjugated via an amino group to a“PEOn” polymer associated with the number n, in general has the formula:CH₃—[—O—CH₂CH₂—]_(n)—C(═O)—NHR, where n is the number of ethylene oxideunits and R denotes the rest of the peptide. The “PEOn” polymer canoptionally have an alkylene group, (CH₂)_(m), where m is an integer from1 to 5, between the carbonyl carbon and the repeating ethylene oxideunits. Such a “PEOn” (e.g., PEO12 or PEO24) polymer is monodispersed,i.e., is a single discrete polymer of a particular molecular weight.Similarly, a CNP peptide conjugated via an amino group to a “PEGnK”polymer associated with the number nK, in general has the formula:CH₃—[—O—CH₂CH₂—]_(p)—C(═O)—NHR, where p is an integer greater than 1.The “PEGnK” polymer also can optionally have an alkylene group,(CH₂)_(m), where m is an integer from 1 to 5, between the carbonylcarbon and the repeating ethylene oxide units. However, such a “PEGnK”(e.g., PEG1K, PEG2K, PEG5K or PEG20K) polymer is polydispersed, i.e.,contains a mixture of polymers having a distribution of molecularweights, where the number nK denotes the polymer number-averagemolecular weight (M_(n)) in kilo Daltons. For example, “PEG2K”conjugated to a CNP peptide denotes a polydispersed PEG polymer having apolymer number-average molecular weight of around 2 kDa.

When a range of the mass of a polymer (e.g., PEG) is given (e.g., inunits of kDa), the range refers to a range of polymer number-averagemolecular weights, not to a range of molecular weights of multiplepolymers in a polydispersed mixture, unless expressly indicatedotherwise.

The term “halogen”, “halide” or “halo” refers to fluorine, chlorine,bromine, and/or iodine.

The term “alkyl” refers to a linear or branched saturated monovalenthydrocarbon radical, wherein the alkyl may optionally be substitutedwith one or more substituents Q as described herein. In certainembodiments, the alkyl is a linear saturated monovalent hydrocarbonradical that has 1 to 20 (C₁₋₂₀), 1 to 15 (C₁₋₁₅), 1 to 12 (C₁₋₁₂), 1 to10 (C₁₋₁₀), or 1 to 6 (C₁₋₆) carbon atoms, or a branched saturatedmonovalent hydrocarbon radical of 3 to 20 (C₃₋₂₀), 3 to 15 (C₃₋₁₅), 3 to12 (C₃₋₁₂), 3 to 10 (C₃₋₁₀), or 3 to 6 (C₃₋₆) carbon atoms. As usedherein, linear C₁₋₆ and branched C₃₋₆ alkyl groups are also referred as“lower alkyl.” Examples of alkyl groups include, but are not limited to,methyl, ethyl, propyl (including all isomeric forms, including n-propyland isopropyl), butyl (including all isomeric forms, including n-butyl,isobutyl, sec-butyl and tert-butyl), pentyl (including all isomericforms), and hexyl (including all isomeric forms). For example, C₁₋₆alkyl refers to a linear saturated monovalent hydrocarbon radical of 1to 6 carbon atoms or a branched saturated monovalent hydrocarbon radicalof 3 to 6 carbon atoms.

The term “alkoxy” refers to an —O-alkyl group. In certain embodiments,an alkoxy group may optionally be substituted with one or moresubstituents Q as described herein.

The term “haloalkyl” refers to an alkyl group that is substituted withone or more halide atoms. In certain embodiments, a haloalkyl group issubstituted with one, two, three, four, five or six halide atoms. Incertain embodiments, a haloalkyl group may optionally be substitutedwith one or more additional substituents Q as described herein.

The term “cycloalkyl” refers to a cyclic saturated bridged and/ornon-bridged monovalent hydrocarbon radical, which may be optionallysubstituted with one or more substituents Q as described herein. Incertain embodiments, the cycloalkyl has from 3 to 20 (C₃₋₂₀), from 3 to15 (C₃₋₁₅), from 3 to 12 (C₃₋₁₂), from 3 to 10 (C₃₋₁₀), or from 3 to 7(C₃₋₇) carbon atoms. Examples of cycloalkyl groups include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, decalinyl, and adamantyl.

The term “heterocyclyl” or “heterocyclic” refers to a monocyclicnon-aromatic ring system or a multicyclic ring system that contains atleast one non-aromatic ring, wherein one or more of the non-aromaticring atoms are heteroatoms independently selected from O, S, or N, andthe remaining non-aromatic ring atoms are carbon atoms. In certainembodiments, the heterocyclyl or heterocyclic group has from 3 to 20,from 3 to 15, from 3 to 10, from 3 to 8, from 4 to 7, or from 5 to 6ring atoms. In certain embodiments, the heterocyclyl is a monocyclic,bicyclic, tricyclic, or tetracyclic ring system, which may include afused or bridged ring system, and in which the nitrogen or sulfur atomsmay be optionally oxidized, the nitrogen atoms may be optionallyquaternized, and some rings may be partially or fully saturated, oraromatic. The heterocyclyl may be attached to the main structure at anyheteroatom or carbon atom which results in the creation of a stablecompound. Examples of heterocyclic groups include, but are not limitedto, acridinyl, azepinyl, benzimidazolyl, benzindolyl, benzoisoxazolyl,benzisoxazinyl, benzodioxanyl, benzodioxolyl, benzofuranonyl,benzofuranyl, benzonaphthofuranyl, benzopyranonyl, benzopyranyl,benzotetrahydrofuranyl, benzotetrahydrothienyl, benzothiadiazolyl,benzothiazolyl, benzothiophenyl, benzotriazolyl, benzothiopyranyl,benzoxazinyl, benzoxazolyl, benzothiazolyl, β-carbolinyl, carbazolyl,chromanyl, chromonyl, cinnolinyl, coumarinyl, decahydroisoquinolinyl,dibenzofuranyl, dihydrobenzisothiazinyl, dihydrobenzisoxazinyl,dihydrofuryl, dihydropyranyl, dioxolanyl, dihydropyrazinyl,dihydropyridinyl, dihydropyrazolyl, dihydropyrimidinyl, dihydropyrrolyl,dioxolanyl, 1,4-dithianyl, furanonyl, furanyl, imidazolidinyl,imidazolinyl, imidazolyl, imidazopyridinyl, imidazothiazolyl, indazolyl,indolinyl, indolizinyl, indolyl, isobenzotetrahydrofuranyl,isobenzotetrahydrothienyl, isobenzothienyl, isochromanyl, isocoumarinyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolidinyl, isothiazolyl,isoxazolidinyl, isoxazolyl, morpholinyl, naphthyridinyl,octahydroindolyl, octahydroisoindolyl, oxadiazolyl, oxazolidinonyl,oxazolidinyl, oxazolopyridinyl, oxazolyl, oxiranyl, perimidinyl,phenanthridinyl, phenathrolinyl, phenarsazinyl, phenazinyl,phenothiazinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,4-piperidonyl, pteridinyl, purinyl, pyrazinyl, pyrazolidinyl, pyrazolyl,pyridazinyl, pyridinyl, pyridopyridinyl, pyrimidinyl, pyrrolidinyl,pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuryl, tetrahydrofuranyl,tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothienyl,tetrazolyl, thiadiazolopyrimidinyl, thiadiazolyl, thiamorpholinyl,thiazolidinyl, thiazolyl, thienyl, triazinyl, triazolyl, and1,3,5-trithianyl. In certain embodiments, a heterocyclic group mayoptionally be substituted with one or more substituents Q as describedherein.

The term “aryl” refers to a monocyclic aromatic group or a multicyclicmonovalent aromatic group that contain at least one aromatic hydrocarbonring. In certain embodiments, the aryl has from 6 to 20 (C₆₋₂₀), from 6to 15 (C₆₋₁₅), or from 6 to 10 (C₆₋₁₀) ring atoms. Examples of arylgroups include, but are not limited to, phenyl, naphthyl, fluorenyl,azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. Arylalso refers to bicyclic or tricyclic carbon rings, where at least one ofthe rings is aromatic and the others may be saturated, partiallyunsaturated, or aromatic, for example, dihydronaphthyl, indenyl,indanyl, and tetrahydronaphthyl (tetralinyl). In certain embodiments, anaryl group may optionally be substituted with one or more substituents Qas described herein.

The term “heteroaryl” refers to a monocyclic aromatic group or amulticyclic aromatic group that contain at least one aromatic ring,wherein at least one aromatic ring contains one or more heteroatomsindependently selected from O, S, and N. Each ring of a heteroaryl groupcan contain one or two O atoms, one or two S atoms, and/or one to four Natoms, provided that the total number of heteroatoms in each ring isfour or less and each ring contains at least one carbon atom. Theheteroaryl may be attached to the main structure at any heteroatom orcarbon atom which results in the creation of a stable compound. Incertain embodiments, the heteroaryl has from 5 to 20, from 5 to 15, orfrom 5 to 10 ring atoms. Examples of monocyclic heteroaryl groupsinclude, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl,imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl,furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl,pyridazinyl, and triazinyl. Examples of bicyclic heteroaryl groupsinclude, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl,benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl,benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl,isobenzofuranyl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl,indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl,dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclicheteroaryl groups include, but are not limited to, carbazolyl,benzindolyl, phenanthrollinyl, acridinyl, phenanthridinyl, andxanthenyl. In certain embodiments, a heteroaryl group may optionally besubstituted with one or more substituents Q as described herein.

The term “optionally substituted” is intended to mean that a group,including alkyl, alkoxy, haloalkyl, cycloalkyl, heterocyclyl, aryl andheteroaryl, may be substituted with one or more substituents Q (in oneembodiment, one, two, three or four substituents Q), where each Q isindependently selected from the group consisting of cyano, halo, oxo,nitro, C₁₋₆ alkyl, C₁₋₆ alkoxy, halo-C₁₋₆ alkyl, C₃₋₇ cycloalkyl,heterocyclyl, C₆₋₁₄ aryl, heteroaryl, —C(O)R^(e), —C(O)OR^(e),—C(O)NR^(f)R^(g), —C(NR^(e))NR^(f)R^(g), —OR^(e), —OC(O)R^(e),—OC(O)OR^(e), —OC(O)NR^(f)R^(g), —OC(═NR^(e))NR^(f)R^(g), —OS(O)R^(e),—OS(O)₂R^(e), —OS(O)NR^(f)R^(g), —OS(O)₂NR^(f)R^(g), —NR^(f)R^(g),—NR^(e)C(O)R^(f), —NR^(e)C(O)OR^(f), —NR^(e)C(O)NR^(f)R^(g),—NR^(e)C(═NR^(h))NR^(f)R^(g), —NR^(e)S(O)R^(f), —NR^(e)S(O)₂R^(f),—NR^(e)S(O)NR^(f)R^(g), —NR^(e)S(O)₂NR^(f)R^(g), —SR^(e), —S(O)R^(e),—S(O)₂R^(e), and —S(O)₂NR^(f)R^(g), wherein each R^(e), R^(f), R^(g),and R^(h) is independently hydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl,heterocyclyl, C₆₋₁₄ aryl, or heteroaryl; or R^(f) and R^(g), togetherwith the N atom to which they are attached, form heterocyclyl.

B. CNP Variants

The use of CNP22 as a therapeutic is limited by its short half-life inplasma (J. Clin. Endocrinol. Metab., 78: 1428-35 (1994)). In humanplasma, the concentration of CNP22 typically is less than fivepicomolar. CNP22 is degraded and cleared from circulation by NEP andNPR-C in humans (Growth Hormone & IGF Res., 16: S6-S14). In all humanand animal studies using systemically administered CNP22, continuousinfusion has been used to increase the CNP22 concentration in thesubjects. A CNP peptide having a longer half-life and at least a similarlevel of functionality would be beneficial to a CNP-based therapeuticstrategy. CNP variants are also disclosed in related InternationalApplication No. PCT/US08/84270, specifically incorporated herein byreference.

The present disclosure provides CNP variants which have reduced affinityto NEP and/or NPR-C, and reduced susceptibility to cleavage by NEPand/or clearance by NPR-C, but which have substantially similar orbetter functionality than wild-type CNP22. Reduced susceptibility of CNPvariants to cleavage by NEP and/or clearance by NPR-C would increase theplasma or serum half-life of the variants, thereby increasing theopportunity for the variants to distribute to the target tissues andsites and effectuate the desired pharmacological effects. In certainembodiments, the CNP variants described herein have reducedsusceptibility to cleavage by NEP and/or clearance by NPR-C in vitro orin vivo by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,4-fold, 4.5-fold, or 5-fold compared to wtCNP22, and have increasedplasma or serum half-life in vivo by at least about 1.5-fold, 2-fold,2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold compared towtCNP22, while retaining at least about 50%, 60%, 70%, 80%, 90% or 100%of the functionality of wtCNP22, or having at least about 1.5-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold greaterfunctionality than wtCNP22. CNP functionality can be evaluated in termsof, e.g., the level of one or more biomarkers (e.g., cGMP) associatedwith cartilage or bone formation or growth in an in vitro or in vivostudy, the length of particular bones in an ex vivo or in vivo study,etc.

Natural substrates of NEP are small and natriuretic peptides (about 2.2to about 3.2 kDa) are the largest of the natural substrates. Accordingto X-ray crystallographic analyses, the NEP active-site is buried deepinside a central cavity, effectively restricting the size of substratemolecules to no more than about 3 kDa (Oefner et al., J. Mol. Biol.,296: 341-349 (2000)). Based on NPR-B signaling studies, variants ofCNP-22, such as CNP-17 (retaining only the cyclic domain, Cys6-Cys22, ofCNP22) and CNP-53 (CNP-22 with a 31-amino acid extension at theN-terminus), can still bind and activate NPR-B similarly to the 2.2 kDawtCNP-22. Accordingly, the disclosure encompasses CNP variantsconjugated to a natural (e.g., peptide) and/or synthetic (e.g., PEG)polymer at the N-terminus and/or C-terminus of CNP22 or variantsthereof, which exhibit increased NEP resistance but retain the abilityto bind and activate the NPR-B signaling receptor.

In one embodiment, the disclosure encompasses CNP variants representedby the general formula:

(x)-Cys₆-Phe₇-Gly₈-Leu₉-Lys₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂-(z)(SEQ ID NO:139), or

(x)-Gly₁-Leu₂-Ser₃-Lys₄-Gly₅-Cys₆-Phe₇-Gly₈-Leu₉-Lys₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂-(z)(SEQ ID NO:140), wherein:

(x) and (z) each independently are a natural polymer (e.g., a peptidesequence containing at least one amino acid) and/or a synthetic polymer(e.g., PEG) as described herein, such that the total mass of the CNPvariant is characterized by the ranges described generally herein, e.g.,in the range from about 2.6 kDa or 2.8 kDa to about 6 or 7 kDa. In oneembodiment, the residues from Cys6 to Cys22 form a cyclic portion. In anembodiment, (x) and/or (z) comprise an amino acid extension derived fromNPPC or a non-CNP polypeptide (e.g., ANP, BNP, IgG, etc.), wherein theextension contains 1 to 40, 1 to 35, 1 to 31, 5 to 35, 5 to 31 or 5 to15 amino acids. In another embodiment, the CNP variants comprise one ormore modifications and/or substitutions with another natural amino acid,an unnatural amino acid, a peptidomimetic and/or a peptide bond isostereat one or more of the following positions of CNP22: Gly1, Lys4, Gly5,Cys6, Phe7, Gly8, Leu9, Lys10, Leu11, Ile14, Gly15, Ser16, Met17, Gly19,Leu20 and Gly21.

In another embodiment, CNP variants having a total mass characterized bythe ranges described generally herein, e.g., from about 2.6 kDa or 2.8kDa to about 6 or 7 kDa, designed for increased resistance to NEPdegradation, are represented by the general formula:

(x)-Cys₆-Phe₇-Gly₈-Leu₉-(h)₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂-(z)(SEQ ID NO: 141), or

(x)-Gly₁-Leu₂-Ser₃-(b)₄-Gly₅-Cys₆-Phe₇-Gly₈-Leu₉-(h)₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂-(z)(SEQ ID NO: 6), wherein:

(x) is a synthetic or natural polymeric group, or a combination thereof,wherein a non-limiting example of a synthetic polymeric group is PEG (orPEO), and a non-limiting example of a natural polymeric group is anamino acid sequence containing from 1 to 35 amino acids and derived fromNPPC or variants thereof with substitutions and/or deletions, ANP, BNP,or other non-CNP (poly)peptides such as, e.g., serum albumin, IgG,histidine-rich glycoproteins, fibronectin, fibrinogen, zincfinger-containing polypeptides, osteocrin or fibroblast growth factor 2(FGF2);

(z) may be absent or may be a synthetic or natural polymeric group, or acombination thereof, wherein a non-limiting example of a syntheticpolymeric group is PEG, and a non-limiting example of a naturalpolymeric group is an amino acid sequence derived from a natriureticpolypeptide (e.g., NPPC, CNP, ANP or BNP) or non-natriuretic polypeptide(e.g., serum albumin or IgG); and

(b) and (h) independently may be the wild type Lys at that position ormay be replaced with a conservative amino acid substitution or anynatural or unnatural amino acid or peptidomimetic that does not have areactive primary amine on a side chain, including but not limited toArg, Gly, 6-hydroxy-norleucine, citrulline (Cit), Gln, Glu or Ser. Inone embodiment, (b) is Arg. In another embodiment, for improved NEPresistance, (b) is not Gly. In yet another embodiment, (h) is not Arg.

Non-limiting examples of amino acid sequences derived from NPPC orvariants thereof include:

Arg, Glu-Arg, (SEQ ID NO: 7) Gly-Ala-Asn-Lys-Lys, (SEQ ID NO: 8)Gly-Ala-Asn-Arg-Arg, (SEQ ID NO: 9) Gly-Ala-Asn-Pro-Arg, (SEQ ID NO: 10)Gly-Ala-Asn-Gln-Gln, (SEQ ID NO: 11) Gly-Ala-Asn-Ser-Ser, (SEQ ID NO:12) Gly-Ala-Asn-Arg-Gln, (SEQ ID NO: 13) Gly-Ala-Asn-Arg-Met, (SEQ IDNO: 14) Gly-Ala-Asn-Arg-Thr, (SEQ ID NO: 15) Gly-Ala-Asn-Arg-Ser, (SEQID NO: 16) Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn- Ala, (SEQ IDNO: 17) Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn- Ala-Arg, (SEQID NO: 18) Asp-Leu-Arg-Val-Asp-Thr-Lys-Ser-Arg-Ala-Ala-Trp- Ala-Arg,(SEQ ID NO: 19) Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Lys-Lys, (SEQ ID NO: 20)Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala- Asn-Arg-Arg, (SEQ IDNO: 21) Asp-Leu-Arg-Val-Asp-Thr-Lys-Ser-Arg-Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Lys-Lys, and (SEQ ID NO: 22)Asp-Leu-Arg-Val-Asp-Thr-Lys-Ser-Arg-Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Arg-Arg.

Non-limiting examples of amino acid sequences derived from non-CNPpolypeptides such as, e.g., ANP, BNP, serum albumin and IgG include:

(SEQ ID NO: 23) Ser-Leu-Arg-Arg-Ser-Ser; (SEQ ID NO: 24)Asn-Ser-Phe-Arg-Tyr; (SEQ ID NO: 25)Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly; (SEQ ID NO: 26)Met-Val-Gln-Gly-Ser-Gly; (SEQ ID NO: 27) Lys-Val-Leu-Arg-Arg-Tyr; (SEQID NO: 28) Lys-Val-Leu-Arg-Arg-His; (SEQ ID NO: 29)Gly-Gln-His-Lys-Asp-Asp-Asn-Pro-Asn-Leu-Pro-Arg; (SEQ ID NO: 30)Gly-Val-Pro-Gln-Val-Ser-Thr-Ser-Thr; (SEQ ID NO: 31)Gly-Glu-Arg-Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg- Leu-Ser-Gln; (SEQ IDNO: 32) Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro- Pro-Ser.

In an embodiment, the N-terminal (x) group and/or the C-terminal (z)group of any of the CNP variants having an (x) and/or (z) group, asdescribed herein, independently comprise an amino acid sequence thatcontains a small number of, if any, acidic natural or unnatural aminoacids (e.g., Asp or Glu). In another embodiment, (x) and/or (z) areenriched in basic natural or unnatural amino acids (e.g., Lys, Arg orHis) to maintain an alkaline pI similar to the pI of CNP22 (pI 8.9). Inone embodiment, the pI of the CNP variants is in the range from about 8to about 10.5, designed so that the CNP variants can diffuse morereadily through the extracellular matrix surrounding chondrocytes ofbone growth plates. In narrower embodiments, the pI of the CNP variantsis from about 8.5 to about 10.5, or from about 8.5 to about 10, or fromabout 9 to about 10.

In yet another embodiment, (x) and/or (z) are enriched in polar naturalor unnatural amino acids, designed for increased aqueous solubility. Instill another embodiment, (x) and/or (z) contain a small number of, ifany, hydrophobic natural or unnatural amino acids (e.g., Ala, Val, Leu,Ile or Met).

In a further embodiment, the N-terminus of the CNP variants terminatesin at least one glycine residue, designed for increased serum half-life.In a related embodiment, to prevent pyroglutamine formation, theN-terminus of CNP variants terminates in a glycine residue if it wouldotherwise terminate in glutamine. In one embodiment, the (x) groupcontains an amino acid extension whose N-terminus terminates in at leastone glycine residue. In another embodiment, (x) and/or (z) do notcontain two adjacent basic natural or unnatural amino acids (e.g.,Lys-Lys or Arg-Arg), designed to reduce susceptibility to cleavage bythe protease furin. In an embodiment, (x) does not contain two adjacentbasic amino acids immediately preceding the position corresponding toGly1 of CNP22.

In still another embodiment, the (x) group and/or the (z) group of theCNP variants comprise an amino acid sequence derived from NPPC (e.g.,derived from CNP53). In an embodiment, (x) comprises an amino acidsequence derived from the N-terminal tail of ANP or BNP. In anotherembodiment, (z) comprises an amino acid sequence derived from theC-terminal tail of ANP or BNP. In a further embodiment, (x) and/or (z)comprise an amino acid sequence derived from a non-natriureticpolypeptide such as, e.g., IgG, human serum albumin (HSA),histidine-rich glycoproteins, fibronectin, fibrinogen, zincfinger-containing polypeptides, FGF-2, and bone-targeting proteins(e.g., osteocrin, osteopontin, osteocalcin, and sialoprotein).

In any embodiment described herein in which CNP22 or a variant thereofcan have an N-terminal (x) group and/or a C-terminal (z) group, (x)and/or (z) independently can contain an amino acid sequence derived fromthe functional domain of a bone morphogenetic protein (BMP). AnN-terminal and/or C-terminal amino acid extension derived from thefunctional domain of a BMP can increase the NEP resistance, and hencethe serum half-life of the CNP variant, by increasing the total mass ofthe CNP variant to characterized by the ranges described generallyherein, e.g., a range from about 2.6 kDa or 2.8 kDa to about 6 or 7 kDa.In addition, since certain BMPs are growth factors and cytokines thatinduce the formation of bone and cartilage, a fragment derived from thefunctional domain of a BMP can promote chondrocyte, cartilage or bonegrowth by a mechanism distinct from activation of the guanylyl cyclasefunction of NPR-B by the cyclic domain of CNP22 or a variant thereof.Non-limiting examples of BMPs that promote bone formation anddevelopment, cartilage formation and development, and/or osteoblastdifferentiation include BMP1, BMP2, BMP3, BMP5, BMP7 and BMP8a. In anembodiment, the N-terminus and/or C-terminus of CNP22 or a variantthereof independently are conjugated to an amino acid sequence derivedfrom the last 140 amino acids in the C-terminal portion of BMP1, BMP2,BMP3, BMP5, BMP7 or BMP8a.

In one embodiment, the CNP variants contain an amino acid extension atthe N-terminus and/or C-terminus of CNP22 or CNP17, including but notlimited to:

(SEQ ID NO: 4) DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC(CNP-53); (SEQ ID NO: 60) QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37,Analog BL); (SEQ ID NO: 61) AAWARLLQEHPNAGLSKGCFGLKLDRIGSMSGLGC (AnalogCA); (SEQ ID NO: 62) AAWARLLQEHPNARGLSKGCFGLKLDRIGSMSGLGC (Analog CB);(SEQ ID NO: 63) DLRVDTKSRAAWARGLSKGCFGLKLDRIGSMSGLGC (Analog CC); (SEQID NO: 40) RGLSKGCFGLKLDRIGSMSGLGC; (SEQ ID NO: 38)ERGLSKGCFGLKLDRIGSMSGLGC; (SEQ ID NO: 64) GANQQGLSKGCFGLKLDRIGSMSGLGC;(SEQ ID NO: 65) GANRRGLSKGCFGLKLDRIGSMSGLGC; (SEQ ID NO: 66)GANPRGLSKGCFGLKLDRIGSMSGLGC; (SEQ ID NO: 67)GANSSGLSKGCFGLKLDRIGSMSGLGC; (SEQ ID NO: 144)GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC; and (SEQ ID NO: 68)SPKMVQGSG-CNP17-KVLRRH (Analog CD) (CNP17 having N-terminal andC-terminal tails derived from BNP).

In another embodiment, the CNP variants have a K4R substitution atposition 4 of CNP22. Non-limiting examples of CNP(K4R) variants include:

(SEQ ID NO: 36) GANRRGLSRGCFGLKLDRIGSMSGLGC (Analog AY); (SEQ ID NO: 37)GANPRGLSRGCFGLKLDRIGSMSGLGC (Analog CI); (SEQ ID NO: 41)RGLSRGCFGLKLDRIGSMSGLGC (Analog AZ); (SEQ ID NO: 39)ERGLSRGCFGLKLDRIGSMSGLGC (Analog BA); (SEQ ID NO: 69)GANQQGLSRGCFGLKLDRIGSMSGLGC (Analog CH); and (SEQ ID NO: 70)GANSSGLSRGCFGLKLDRIGSMSGLGC (Analog CG).

In further embodiments, the CNP variants are chimeras comprising CNP22,or a variant thereof having amino acid addition(s), deletion(s) and/orsubstitution(s), and a peptide fragment derived from a polypeptide orprotein other than CNP, or the whole non-CNP polypeptide or protein, tothe N-terminus of the CNP peptide, wherein CNP22 or the variant thereofmay optionally have an N-terminal amino acid extension of one or moreamino acid residues. In certain embodiments, the CNP chimeras compriseCNP22 or a variant thereof that has an N-terminal amino acid extensionof one or more amino acid residues. In certain embodiments, the CNPchimeras contain lysine-lysine (KK) residues or GANKK residuesimmediately preceding the first position of CNP22 (Gly in the case ofCNP22) or a variant thereof. In other embodiments, the CNP chimerascontain one or two residues different from lysine-lysine immediatelypreceding the first position of CNP22 or a variant thereof. Non-limitingexamples of residues that can immediately precede the first position ofCNP22 or a variant thereof include KP, PK, PR, PQ, QK, QQ, RR, SS,GANKP(SEQ ID NO: 200), GANPK (SEQ ID NO: 201), GANPR (SEQ ID NO: 9),GANPQ (SEQ ID NO: 202), GANQK (SEQ ID NO: 203), GANQQ (SEQ ID NO: 10),GANRR (SEQ ID NO: 8), and GANSS (SEQ ID NO: 11).

In another embodiment, the CNP variants are chimera comprising CNP22 andan N-terminal peptide fragment, including but not limited to:

(SEQ ID NO: 76) GHHSHEQHPHGANQQGLSKGCFGLKLDRIGSMSGLGC (Analog CQ)(histidine-rich glycoprotein (HRGP) fragment-CNP22 chimera);(SEQ ID NO: 77) GAHHPHEHDTHGANQQGLSKGCFGLKLDRIGSMSGLGC (Analog CR)(HRGP fragment-CNP22 chimera); (SEQ ID NO: 78)GHHSHEQHPHGANPRGLSKGCFGLKLDRIGSMSGLGC (Analog CX)(HRGP fragment-CNP22 chimera); (SEQ ID NO: 79)GQPREPQVYTLPPSGLSKGCFGLKLDRIGSMSGLGC (Analog CF)(IgG₁(F_(c)) fragment-CNP22 chimera); (SEQ ID NO: 80)GQHKDDNPNLPRGANPRGLSKGCFGLKLDRIGSMSGLGC(Analog CY) (human serum albumin (HSA) fragment- CNP22 chimera);(SEQ ID NO: 81) GERAFKAWAVARLSQGLSKGCFGLKLDRIGSMSGLGC(Analog CE) (HSA fragment-CNP22 chimera); (SEQ ID NO: 82)FGIPMDRIGRNPRGLSKGCFGLKLDRIGSMSGLGC (Analog CZ)(osteocrin “NPR C inhibitor” fragment-CNP22 chimera); and(SEQ ID NO: 83) GKRTGQYKLGSKTGPGPKGLSKGCFGLKLDRIGSMSGLGC(Analog DA) (FGF2 “heparin-binding domain” fragment-CNP22 chimera).

In yet another embodiment, the CNP variants are chimera comprising anN-terminal peptide fragment and CNP22 in which arginine is substitutedfor Lys4 of CNP22 (“CNP22(K4R)”), including but not limited to:

(SEQ ID NO: 84) GQPREPQVYTGANQQGLSRGCFGLKLDRIGSMSGLGC (Analog CK)(IgG₁(F_(c)) fragment-CNP22(K4R) chimera); (SEQ ID NO: 85)GVPQVSTSTGANQQGLSRGCFGLKLDRIGSMSGLGC (Analog CL)(HSA fragment-CNP22(K4R) chimera); (SEQ ID NO: 86)GQPSSSSQSTGANQQGLSRGCFGLKLDRIGSMSGLGC (AnalogCM) (fibronectin fragment-CNP22(K4R) chimera); (SEQ ID NO: 87)GQTHSSGTQSGANQQGLSRGCFGLKLDRIGSMSGLGC (AnalogCN) (fibrinogen fragment-CNP22(K4R) chimera); (SEQ ID NO: 88)GSTGQWHSESGANQQGLSRGCFGLKLDRIGSMSGLGC (AnalogCO) (fibrinogen fragment-CNP22(K4R) chimera); and (SEQ ID NO: 89)GSSSSSSSSSGANQQGLSRGCFGLKLDRIGSMSGLGC (AnalogCP) (zinc finger fragment-CNP22(K4R) chimera).

Chimera comprising IgG and CNP22 or a variant thereof are designed for,inter alia, increased resistance to NEP degradation and reduced bindingto serum albumin. CNP chimera comprising a surface fragment of HSA aredesigned for, inter alia, reduced immunogenicity and reduced binding toserum albumin. HRGP-CNP22 and HRGP-CNP22(K4R) chimera containing acationic, histidine-rich, non-lysine, non-arginine sequence at theN-terminus are designed for, inter alia, increased stability toproteases. Chimera containing an osteocrin fragment are designed torelease, upon protease (e.g., furin) cleavage, the osteocrin fragment atbone growth plates, where the fragment would inhibit the clearancereceptor NPR-C. With respect to chimera comprising an FGF2heparin-binding fragment, heparin binding to the fragment is designed toprotect the chimera from degradation, thereby providing a longer serumhalf-life. Chimera containing a fibronectin, fibrinogen, or zinc-fingerfragment are designed for reduced binding to serum albumin, among otherfeatures.

Not intending to be bound by theory, a CNP variant of molecular weightfrom about 2.6 or 2.8 kDa to about 6 or 7 kDa which has increasedresistance to NEP degradation and has similar or improved functionality(e.g., binding to NPR-B and stimulation of cGMP signaling) as comparedto wtCNP22, may be more effective if it does not bind tightly to plasmaproteins such as serum albumin. A CNP variant that does not bind tightlyto plasma proteins (e.g., serum albumin) may be more effective indiffusing through cartilage, getting to chondrocytes of bone growthplates, and binding to and activating NPR-B for cGMP signaling. In oneembodiment, CNP variants designed for reduced binding to plasma proteins(e.g., serum albumin) are chimeras comprising CNP22 or a variant thereofand a peptide fragment from IgG. In another embodiment, CNP variantsdesigned for reduced binding to plasma proteins are chimeras comprisingCNP22 or CNP22(K4R) and a fragment from a polypeptide (e.g., IgG, HSA,fibronectin, fibrinogen, a zinc finger-containing polypeptide, etc.). Inyet another embodiment, CNP variants designed for reduced binding toplasma proteins comprise CNP22 or a variant thereof conjugated to ahydrophilic or water-soluble polymer. In one embodiment, the hydrophilicor water-soluble polymer is PEG (or PEO). In another embodiment, thehydrophilic or water-soluble polymer (e.g., PEG) is functionalized withone or more functional groups that impart a negative charge to thepolymer under physiological conditions, such as, e.g, carboxyl, sulfateor phosphate groups, or a combination thereof.

In a further embodiment, CNP variants of the disclosure includetruncated CNP peptides ranging from human CNP-17 (hCNP-17) to humanCNP-53 (hCNP-53), and having wild-type amino acid sequences derived fromhCNP-53. Such truncated CNP peptides include:

(SEQ ID NO: 4) DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-53); (SEQ ID NO: 146)LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGL GC (CNP-52);(SEQ ID NO: 147) RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-51); (SEQ ID NO: 148)VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-50);(SEQ ID NO: 149) DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-49); (SEQ ID NO: 150)TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-48);(SEQ ID NO: 151) KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47); (SEQ ID NO: 152)SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-46);(SEQ ID NO: 153) RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-45);(SEQ ID NO: 154) AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-44);(SEQ ID NO: 155) AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-43);(SEQ ID NO: 156) WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-42);(SEQ ID NO: 157) ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-41);(SEQ ID NO: 158) RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-40);(SEQ ID NO: 159) LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-39);(SEQ ID NO: 160) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38);(SEQ ID NO: 60) QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37);(SEQ ID NO: 161) EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-36);(SEQ ID NO: 162) HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-35);(SEQ ID NO: 163) PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34);(SEQ ID NO: 164) NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-33);(SEQ ID NO: 165) ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-32);(SEQ ID NO: 166) RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-31);(SEQ ID NO: 167) KYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-30);(SEQ ID NO: 168) YKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-29);(SEQ ID NO: 169) KGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-28); (SEQ ID NO: 170)GANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-27); (SEQ ID NO: 171)ANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-26); (SEQ ID NO: 172)NKKGLSKGCFGLKLDRIGSMSGLGC (CNP-25); (SEQ ID NO: 173)KKGLSKGCFGLKLDRIGSMSGLGC (CNP-24); (SEQ ID NO: 174)KGLSKGCFGLKLDRIGSMSGLGC (CNP-23); (SEQ ID NO: 1)GLSKGCFGLKLDRIGSMSGLGC (CNP-22); (SEQ ID NO: 175)LSKGCFGLKLDRIGSMSGLGC (CNP-21); (SEQ ID NO: 176)SKGCFGLKLDRIGSMSGLGC (CNP-20); (SEQ ID NO: 177)KGCFGLKLDRIGSMSGLGC (CNP-19); (SEQ ID NO: 178)GCFGLKLDRIGSMSGLGC (CNP-18); and (SEQ ID NO: 2)CFGLKLDRIGSMSGLGC (CNP-17).In certain embodiments, CNP variants do not include CNP-17, CNP-22 orCNP-53.

In another embodiment, the truncated CNP peptides ranging from hCNP-17to hCNP-53 can contain amino acid addition(s), deletion(s) and/orsubstitution(s) with natural or unnatural amino acid(s) orpeptidomimetic(s) (e.g., peptide bond isostere(s)), as described herein,at any one or more of the amino acid positions of the particulartruncated CNP peptides. In yet another embodiment, the truncated CNPpeptides having wild-type sequences or amino acid addition(s),deletion(s) and/or substitution(s), can be conjugated at the N-terminus,C-terminus and/or internal site(s) to any of the moieties describedherein, including but not limited to bone- or cartilage-targetingmoieties (e.g., bisphosphonates, bone- or cartilage-targeting peptidesequences (e.g., polyAsp, polyGlu), peptide sequences derived frombone-targeting domains of bone proteins (e.g., osteopontin, osteocalcin,sialoprotein)), peptide sequences derived from the functional domains ofbone morphogenetic proteins (e.g., BMP2, BMP3, BMP5, BMP7, BMP8a),peptide sequences derived from natriuretic polypeptides (e.g., NPPC,ANP, BNP), peptide sequences derived from polypeptides ofnon-natriuretic origin (e.g., serum albumin, IgG, histidine-richglycoproteins, fibronectin, fibrinogen, zinc finger-containingpolypeptides, FGF-2, osteocrin), moieties that reduce renal clearance(e.g., negatively charged PEG moieties), hydrophilic polymers (e.g.,PEG), carbohydrates (e.g., carbohydrates recognized by receptors on thesurface of cells at bone growth plates), hydrophobic acids (e.g., C₅-C₁₂carboxylic acids, natural fatty acids), phospholipids, and combinationsthereof. In an embodiment, the truncated CNP peptides having wild-typesequences or amino acid addition(s), deletion(s) and/or substitution(s),and optionally conjugated to one or more moieties at the N-terminus,C-terminus and/or internal site(s), have a total mass characterized bythe ranges described generally herein, e.g., from about 2.6 kDa or 2.8kDa to about 6 or 7 kDa.

In a further embodiment, the CNP variants are derivatives of CNP37,which is QEHPNARKYKGANKK-CNP22 (SEQ ID NO: 60). The CNP37 variants cancontain amino acid addition(s), deletion(s), and/or substitution(s) withnatural or unnatural amino acid(s) or peptidomimetic(s) (e.g., peptidebond isostere(s)) at any one or more of the 37 positions of CNP37.Non-limiting examples of substitutions that can be made in CNP37, basedon the numbering of CNP22, include K4R, G5S, G5R, G8S, K10R, G15S, S16Q,M17N, G19R, and combinations thereof. In an embodiment, the CNP37derivatives contain a substitution of Met17 to a natural (e.g.,asparagine) or unnatural amino acid or peptidomimetic, designed in partto avoid oxidation of the sulfur atom of methionine. In anotherembodiment, the CNP37 variants contain substitution(s) of Lys8, Lys10,Lys14 and/or Lys15 (based on numbering from the N-terminus of CNP37) tonon-basic natural or unnatural amino acid(s) or peptiomimetic(s),designed in part to reduce albumin binding.

In addition or alternatively to amino acid addition(s), deletion(s)and/or substitution(s), the CNP37 derivatives can be conjugated at theN-terminus, C-terminus, and/or an internal site to any of the moietiesdescribed herein, including but not limited to bone- orcartilage-targeting moieties (e.g., bone-targeting peptide domains),moieties that reduce renal clearance (e.g., negatively charged PEGmoieties), hydrophilic polymers (e.g., PEG), amino acid sequencescomprising one or more amino acids (e.g., osteocrin “NPR-C inhibitor”fragment), carbohydrates (e.g., carbohydrates recognized by receptors onthe surface of cells at bone growth plates), hydrophobic acids (e.g.,C₅-C₁₂ carboxylic acids and natural fatty acids), and combinationsthereof.

In one embodiment, the CNP variants are modified CNP37 peptides havingmutation(s)/substitution(s) at the furin cleavage site (underlined),designed to improve in vivo resistance to the furin protease, and/orcontaining glycine (underlined) at the N-terminus, designed to improveplasma stability and prevent pyroglutamine formation. Such CNP37variants include but are not limited to:

(SEQ ID NO: 71) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (An. CS);(SEQ ID NO: 72) GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (An. CT);(SEQ ID NO: 73) GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (An. CU);(SEQ ID NO: 74) GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (An. CW);(SEQ ID NO: 75) GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP37, An. DB); and (SEQ ID NO: 145)PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP37)

In a further embodiment, the CNP variants of the disclosure include CNPpeptides and variants thereof that can be produced by the fusion proteinprocess described herein. Non-limiting examples of CNP variants that canbe produced by the fusion protein process described herein, usingchemical or proteolytic cleavage or protein self-cleavage, include:

(SEQ ID NO: 179) GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-wtCNP53); (SEQ ID NO: 75)GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-wtCNP37); (SEQ ID NO: 60)QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (wtCNP37); (SEQ ID NO: 144)GHKSEVAHRFK-GANKKGLSKGCFGLKLDRIGSMSGLGC (HSA fragment-wtCNP27);(SEQ ID NO: 36) GANRRGLSRGCFGLKLDRIGSMSGLGC [CNP27(K4,5,9R)];(SEQ ID NO: 180) DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP53(M48N)]; (SEQ ID NO: 181)GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP37(M32N)];(SEQ ID NO: 182) QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP37(M32N)];(SEQ ID NO: 183) GHKSEVAHRFK-GANKKGLSKGCFGLKLDRIGSNSGLGC[HSA-CNP27(M22N)]; (SEQ ID NO: 184)GANRRGLSRGCFGLKLDRIGSNSGLGC [CNP27(K4,5,9R, M22N)]; (SEQ ID NO: 185)PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC (Pro-wtCNP53);(SEQ ID NO: 145) PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-wtCNP37); (SEQ ID NO: 186)PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-wtCNP37); (SEQ ID NO: 187)PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (wtCNP34); (SEQ ID NO: 188)P-GHKSEVAHRFK-GANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-HSA-wtCNP27);(SEQ ID NO: 189) PGANRRGLSRGCFGLKLDRIGSMSGLGC [Pro-CNP27(K4,5,9R)];(SEQ ID NO: 190) MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-wtCNP53); (SEQ ID NO: 191)MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-wtCNP37);(SEQ ID NO: 192) MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-wtCNP37);(SEQ ID NO: 193) M-GHKSEVAHRFK-GANKKGLSKGCFGLKLDRIGSMSGLGC(Met-HSA-wtCNP27); (SEQ ID NO: 194)MGANRRGLSRGCFGLKLDRIGSMSGLGC [Met-CNP27(K4,5,9R)].

Other CNP variants, including truncated CNP peptides ranging fromhCNP-17 to hCNP-53 and having wild-type sequences or amino acidaddition(s), deletion(s) and/or substitution(s), can also be produced bythe fusion protein process described herein, so long as the intendedsite of chemical or proteolytic cleavage of the fusion protein is notpresent within the amino acid sequence of the target CNP variant itself.As a non-limiting example, the fusion protein process described hereincan be employed to produce truncated wtCNP34 using formic acid cleavage.

In additional embodiments, for any of the CNP peptides and CNP variantsdescribed herein that have asparagine (Asn/N) residue(s) and/orglutamine (Gln/Q) residue(s), whether they have a wild-type sequence ora non-natural amino acid sequence, any Asn residue(s) and/or any Glnresidue(s) can independently be substituted with any other natural orunnatural amino acids, including conservative substitutions such as Asnto Gln. Such substitution(s) are designed in part to minimize or avoidany potential deamidation of asparagine and/or glutamine. Non-limitingexamples of CNP peptides and variants in which any Asn residue(s) and/orany Gln residue(s) can independently be substituted with any othernatural or unnatural amino acids, including conservative substitutionssuch as Asn to Gln, include wtCNP34, wtCNP37, Gly-wtCNP37, Pro-wtCNP37,Pro-Gly-wtCNP37, GHKSEVAHRFK-wtCNP27 (SEQ ID NO: 144),Pro-GHKSEVAHRFK-wtCNP27 (SEQ ID NO: 188), PEO12-GANRR-CNP22(K4R) (SEQ IDNO: 36), and PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36). In certainembodiments, an asparagine residue of the CNP peptides and CNP variantsdescribed herein is not substituted with glutamine, aspartic acid orglutamic acid. In certain embodiments, a glutamine residue of the CNPpeptides and CNP variants described herein is not substituted withasparagine, aspartic acid or glutamic acid. As a non-limiting example,asparagine residues 7 and/or 15 of Pro-Gly-wtCNP37(PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC) (SEQ ID NO: 145) canindependently be substituted with any other natural or unnatural aminoacids, including glutamine, to avoid any potential deamidation of theasparagine residue(s) to aspartic acid or isoaspartic acid. In certainembodiments, asparagine residues 7 and/or 15 of Pro-Gly-wtCNP37 are notsubstituted with glutamine, aspartic acid or glutamic acid.

It is understood, however, that the present disclosure encompasses CNPvariants in which any one or more, up to all, residues susceptible todeamidation or a deamidation-like reaction (e.g., isomerization) may beconverted to other residue(s) via deamidation or a deamidation-likereaction to any extent, up to 100% conversion per converted residue. Incertain embodiments, the disclosure encompasses CNP variants in which:

-   (1) any one or more, up to all, asparagine (Asn/N) residues may be    converted to aspartic acid or aspartate, and/or to isoaspartic acid    or isoaspartate, via deamidation up to about 5%, 10%, 20%, 30%, 40%,    50%, 60%, 70%, 80%, 90% or 100% conversion per converted residue; or-   (2) any one or more, up to all, glutamine (Gln/Q) residues may be    converted to glutamic acid or glutamate, and/or to isoglutamic acid    or isoglutamate, via deamidation up to about 5%, 10%, 20%, 30%, 40%,    50%, 60%, 70%, 80%, 90% or 100% conversion per converted residue; or-   (3) any one or more, up to all, aspartic acid or aspartate (Asp/D)    residues may be converted to isoaspartic acid or isoaspartate via a    deamidation-like reaction (also called isomerization) up to about    5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% conversion    per converted residue; or-   (4) any one or more, up to all, glutamic acid or glutamate (Glu/E)    residues may be converted to isoglutamic acid or isoglutamate via a    deamidation-like reaction (also called isomerization) up to about    5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% conversion    per converted residue; or-   (5) a combination of the above.

As a non-limiting example, the disclosure encompasses CNP variants inwhich any one or more, up to all, asparagine, glutamine, aspartic acid,and/or glutamic acid residues of Pro-Gly-wtCNP37[PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC] (SEQ ID NO: 145) may beconverted to (1) aspartic acid/aspartate and/or isoasparticacid/isoaspartate, (2) glutamic acid/glutamate and/or isoglutamicacid/isoglutamate, (3) isoaspartic acid/isoaspartate, and/or (4)isoglutamic acid/isoglutamate, respectively, via deamidation or adeamidation-like reaction up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100% conversion per converted residue, as describedabove.

As a further example, the disclosure encompasses CNP variants in whichany one or more, up to all, asparagine and/or aspartic acid residues ofPro-Gly-wtCNP37 [PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC] (SEQ ID NO:145) may be converted to (1) aspartic acid/aspartate and/or isoasparticacid/isoaspartate, and/or (2) isoaspartic acid/isoaspartate,respectively, via deamidation or a deamidation-like reaction up to about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% conversion perconverted residue.

As another example, the present disclosure encompasses CNP variants inwhich any one or more, up to all, asparagine, glutamine, aspartic acid,and/or glutamic acid residues of Gly-wtCNP37[GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 75)] may beconverted to (1) aspartic acid/aspartate and/or isoasparticacid/isoaspartate, (2) glutamic acid/glutamate and/or isoglutamicacid/isoglutamate, (3) isoaspartic acid/isoaspartate, and/or (4)isoglutamic acid/isoglutamate, respectively, via deamidation or adeamidation-like reaction up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100% conversion per converted residue.

As yet another example, the disclosure encompasses CNP variants in whichany one or more, up to all, asparagine, glutamine, aspartic acid, and/orglutamic acid residues of wtCNP37 [QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO: 60)] may be converted to (1) aspartic acid/aspartate and/orisoaspartic acid/isoaspartate, (2) glutamic acid/glutamate and/orisoglutamic acid/isoglutamate, (3) isoaspartic acid/isoaspartate, and/or(4) isoglutamic acid/isoglutamate, respectively, via deamidation or adeamidation-like reaction up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100% conversion per converted residue.

As a further example, the present disclosure encompasses CNP variants inwhich any one or more, up to all, asparagine, aspartic acid, and/orglutamic acid residues of an HSA-wtCNP27 chimera,GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 144), may beconverted to (1) aspartic acid/aspartate and/or isoasparticacid/isoaspartate, (2) isoaspartic acid/isoaspartate, and/or (3)isoglutamic acid/isoglutamate, respectively, via deamidation or adeamidation-like reaction up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100% conversion per converted residue.

As a still further example, the disclosure encompasses CNP variants inwhich any one or more, up to all, asparagine, aspartic acid, and/orglutamic acid residues of a Pro-HSA-wtCNP27 chimera,PGHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 188), may beconverted to (1) aspartic acid/aspartate and/or isoasparticacid/isoaspartate, (2) isoaspartic acid/isoaspartate, and/or (3)isoglutamic acid/isoglutamate, respectively, via deamidation or adeamidation-like reaction up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100% conversion per converted residue.

In addition, the present disclosure encompasses CNP variants in whichany one or more, up to all, methionine (Met/M) residues may be oxidizedto any chemically feasible oxidized form (e.g., sulfoxide and/orsulfone) up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or100% transformation per oxidized residue.

In another embodiment, the CNP variants comprise CNP22 or variantsthereof conjugated at the N-terminus and/or C-terminus to moiet(ies)that facilitate translocation of the variants across a cell membrane orcell barrier. In one embodiment, the CNP variants are conjugated at theN-terminus and/or C-terminus to peptide sequence(s) that facilitatetransport of the variants across a cell membrane or cell barrier,including via active peptide transporters.

In a further embodiment, the N-terminus and/or C-terminus of CNP22 or avariant thereof are conjugated to chemical moieties such as, e.g.,natural and/or synthetic polymers, to increase the total mass of themodified CNP peptide to the ranges described generally herein, e.g., arange from about 2.6 or 2.8 kDa to about 6 or 7 kDa. In one embodiment,the chemical moieties are biocompatible hydrophilic or water-solublenatural (e.g., peptides, carbohydrates) or synthetic (e.g., PEG (orPEO)) polymers.

In a particular embodiment, the N-terminus and/or C-terminus of CNP22 ora variant thereof are conjugated to PEG (or PEO) polymers to result in atotal mass characterized by the ranges described generally herein, e.g.,from about 2.6 or 2.8 kDa to about 6 or 7 kDa. Pegylation of CNP22 or avariant thereof is designed, inter alia, to reduce immunogenicity andimprove half-life by reducing renal clearance and increasing proteaseresistance. A PEG moiety can be attached to the N- and/or C-terminus ofCNP22 or any variant described herein, including but not limited toCNP-17 (the Cys6-Cys22 cyclized portion of CNP22), CNP37, and variantsof CNP17, CNP22 or CNP37 having N- and/or C-terminal amino acidextension(s), amino acid substitution(s) and/or amino acid deletion(s).In an embodiment, the Lys4 and/or Lys10 residues of CNP17, CNP22 orCNP37, or variants thereof, are substituted with a natural or unnaturalamino acid (e.g., Arg, Gly, Ser, Gln, Glu or Cit) or peptidomimetic thatdoes not contain a reactive primary amine on a side chain, to precludeany potential PEGylation of these lysine residues. In one embodiment,the Lys4 and/or Lys10 residues of the CNP peptides are substituted withArg. In another embodiment, the Lys10 residue is not substituted withArg.

In a further embodiment, CNP variants (including CNP22 and variantsthereof) having a PEG (or PEO) moiety and an amino acid extension at theN-terminus contain arginine at the position immediately preceding theposition corresponding to Gly1 of CNP22. Such PEGylated CNP variants aredesigned for increased resistance to NEP degradation, reduced binding toserum albumin, and enhanced CNP functional activity (e.g., activation ofcGMP signaling). Non-limiting examples of PEGylated CNP variants includePEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36), PEO12-GANRR-CNP22(K4R) (SEQ IDNO: 36), PEO24-GANRR-CNP22(SEQ ID NO: 36), PEO12-GANRR-CNP22(SEQ ID NO:36), PEO24-GANPR-CNP22(K4R) (SEQ ID NO: 37), PEO12-GANPR-CNP22(K4R) (SEQID NO: 37), PEO24-GANPR-CNP22(SEQ ID NO: 37), PEO12-GANPR-CNP22(SEQ IDNO: 37), PEO24-GANQQ-CNP22(SEQ ID NO: 64), PEO12-GANQQ-CNP22(SEQ ID NO:64), PEO24-ER-CNP22(K4R) (SEQ ID NO: 39), PEO12-ER-CNP22(K4R) (SEQ IDNO: 39), PEO24-ER-CNP22(SEQ ID NO: 39), PEO12-ER-CNP22(SEQ ID NO: 39),PEO24-R-CNP22(K4R) (SEQ ID NO: 41), PEO12-R-CNP22(K4R) (SEQ ID NO: 41),PEO24-R-CNP22(SEQ ID NO: 41), and PEO12-R-CNP22(SEQ ID NO: 41), whereinPEO24 is a monodispersed 1.2 kDa PEG polymer and PEO12 is amonodispersed 0.6 kDa PEG polymer. In one embodiment, the PEG (or PEO)polymer is conjugated to the N-terminus of the CNP variants.

The disclosure contemplates use of hydrophilic or water soluble polymers(e.g., PEG) that can vary in type (e.g., homopolymer or copolymer;random, alternating or block copolymer; linear or branched;monodispersed or polydispersed), linkage (e.g., hydrolysable or stablelinkage such as, e.g., amide, imine, aminal, alkylene, or ester bond),conjugation site (e.g., at the N-terminus and/or C-terminus, preferablynot at any of the residues in the cyclized region of CNP (correspondingto residues 6-22 of CNP22)), and length (e.g., from about 0.2, 0.4 or0.6 kDa to about 2, 3, 4 or 5 kDa). The hydrophilic or water-solublepolymer can be conjugated to the CNP peptide by means of N-hydroxysuccinimide (NHS)- or aldehyde-based chemistry or other chemistry, as isknown in the art. Such CNP variants can be generated using, e.g.,wtCNP22 (2.2 kDa), CNP17 retaining only the cyclized region (residues6-22) of wtCNP22, CNP variants having an amino acid extension at theN-terminus and/or C-terminus of CNP22 or CNP17, or variants having aminoacid substitutions, additions and/or deletions such as, e.g.,GANRR-CNP22(K4R) (SEQ ID NO: 36), GANPR-CNP22(K4R) (SEQ ID NO: 37),R-CNP22(SEQ ID NO: 40), R-CNP22(K4R) (SEQ ID NO: 41), ER-CNP22(SEQ IDNO: 38) and ER-CNP22(K4R) (SEQ ID NO: 39). In an embodiment, the PEG-CNPvariants having a total mass characterized by the ranges describedgenerally herein, e.g., from about 2.6 or 2.8 kDa to about 6 or 7 kDa,contain a monodispersed, linear PEG (or PEO) moiety conjugated at theN-terminus and/or C-terminus via NHS- or aldehyde-based chemistry, or atwo-arm or three-arm branched PEG moiety conjugated at the N-terminusand/or C-terminus via NHS-based chemistry. The disclosure alsoencompasses negatively charged PEG-CNP variants designed for reducedrenal clearance, including but not limited to carboxylated, sulfated andphosphorylated compounds (Caliceti, Adv. Drug Deliv. Rev., 55: 1261-77(2003); Perlman, J. Clin. Endo. Metab., 88: 3227-35 (2003); Pitkin,Antimicrob. Ag. Chemo., 29: 440-444 (1986); Vehaskari, Kidney Int'l, 22:127-135 (1982)). In one embodiment, the PEG (or PEO) moiety containscarboxyl group(s), sulfate group(s), and/or phosphate group(s).

In another embodiment, the PEG (or PEO) moieties conjugated to theN-terminus, C-terminus and/or internal site(s) of CNP variants describedherein contain one or more functional groups that are positively chargedunder physiological conditions. Such PEG moieties are designed, interalia, to improve distribution of such PEGylated CNP variants tocartilage tissues. In one embodiment, such PEG moieties contain one ormore primary, secondary or tertiary amino groups, quaternary ammoniumgroups, and/or other amine-containing (e.g., urea) groups.

In an embodiment, the disclosure encompasses CNP22 or variants thereofconjugated via NHS- or aldehyde-based chemistry to PEG (or PEO) of theformula (CH₂CH₂O)_(n), wherein n is an integer from about 6 to about100, and the PEG polymer is from about 0.3 kDa to about 5 kDa. Inanother embodiment, n is an integer from about 12 to about 50, and thePEG polymer is from about 0.6 kDa to about 2.5 kDa. In yet anotherembodiment, n is from about 12 to about 24, and the PEG polymer is fromabout 0.6 kDa to about 1.2 kDa. In still another embodiment, theterminal hydroxyl group of the PEG polymer is capped with a non-reactivegroup. In a particular embodiment, the end-capping group is an alkylgroup, e.g., a lower alkyl group such as methyl.

In a further embodiment, the disclosure provides CNP variants having oneor more peptide bonds or peptide bond isosteres that have reducedsusceptibility to cleavage by peptidases including neutral endopeptidase(NEP). NEP is a membrane-bound zinc-dependent endopeptidase that cleavesa substrate peptide bond at the amino end of large hydrophobic residues.Thus, modification of a peptide bond at a cleavage site for NEP to anunnatural peptide or non-peptide bond may preclude or decrease theefficiency of NEP cleavage.

For ANP and CNP, NEP cleavage is reported to occur first at theCys6-Phe7 bond within the cyclized region, then elsewhere throughout theremainder of the structures. For BNP, cleavage is reported to occurfirst at the peptide N-terminus, then within the cyclic structure.Although the primary NEP cleavage site on CNP is reported to be theCys6-Phe7 bond, when wtCNP22 was exposed to NEP digestion for 2.5minutes in vitro, all possible sites were unexpectedly hydrolyzed, withthe Cys6-Phe7 and Gly8-Leu9 peptide bonds being slightly most labile, asdescribed in Example 2.

Substrate specificity of NEP is primarily determined by twosubstrate-binding subsites, S1′ and S2′ (Oefner et al., J. Mol. Biol.296:341-349 (2000)). The S1′ site accepts a large hydrophobic P1′residue of which the N-terminal peptide bond is subjected to hydrolysis(e.g., Phe, Leu, Be and Met). The S2′ site generally prefers a smallerresidue, termed P2′ (e.g., Gly or Ser). In the case of CNP, Phe7 isreported to be the preferred P1′ residue for the NEP S1′ site, whileGly8 is the preferred P2′ residue for the S2′ site. Because these twosubsites can together accommodate only a certain total side chain size,any increase in the total size of the P1′-P2′ residues of CNP canpotentially disrupt NEP binding. For example, addition of a chlorideatom at the 3-position of the P1′ Phe7 aromatic ring (i.e., 3-Cl-Phe7)can potentially modify (e.g., destabilize) interactions between CNP andthe NEP cleavage sites, for example at the S1′ subsite. Addition of atertiary butyl group to the smaller P2′ residue Gly8 (i.e., tBu-Gly8)can potentially disrupt the interaction between CNP and the S2′ subsite.

Accordingly, in one embodiment, CNP variants of the disclosure includeCNP having an increase in the size of the P1′-P2′ residues, such asPhe7-Gly8, to interfere with substrate recognition at the active site,thereby reducing susceptibility to NEP cleavage. Natural amino acids,unnatural amino acids and/or peptidomimetic moieties are substituted forone or more large P1′ hydrophobic residues, including but not limited toPhe7, Leu9, Leu11, Ile14, Met17 and Leu20, and/or for one or moresmaller P2′ residues, including but not limited to Cys6, Gly8, Gly15,Ser16 and Gly19.

The disclosure encompasses CNP variants comprising at least one modifiedamino acid and/or at least one modified peptide bond, at at least oneresidue involved in substrate recognition and/or cleavage by NEP,wherein the modified amino acids and modified peptide bonds can benatural amino acids, unnatural amino acids, peptidomimetics and/orpeptide bond isosteres. In one embodiment, the NEP cleavage site on CNPbetween Cys6 and Phe7 is modified. In a related embodiment, the peptidebond (—C(═O)—NH—) between Cys6 and Phe7 is replaced with one of thefollowing peptide bond isosteres:

—CH₂—NH—,

—C(═O)—N(R)—, where the amide group is alkylated with any of thefollowing R groups: methyl, ethyl, n-propyl, isopropyl, cyclopropyl,n-butyl, isobutyl, sec-butyl or tert-butyl,

—C(═O)—NH—CH₂—,

—CH₂—S—,

—CH₂—S(O)_(n)—, where n is 1 or 2,

—CH₂—CH₂—,

—CH═CH—,

—C(═O)—CH₂—,

—CH(CN)—NH—,

—CH(OH)—CH₂—,

—O—C(═O)—NH—, or

—NHC(═O)NH—.

In another embodiment, the CNP variants are represented by the formula:

-   (x)-Gly₁-Leu₂-Ser₃-Lys₄-Gly₅-(b)₆-(c)₇-(d)₈-Leu₉-Lys₁₀-Leu₁₁-Asp₁₂-Arg₁₃-Ile₁₄-Gly₁₅-Ser₁₆-Met₁₇-Ser₁₈-Gly₁₉-Leu₂₀-Gly₂₁-Cys₂₂-(z)    (SEQ ID NO: 90), wherein:

(x) and (z) independently may be absent or may be selected from thegroup consisting of synthetic bone-targeting compounds such as, e.g.,bisphosphonates; amino acid sequences useful in bone or cartilagetargeting such as, e.g., polyAsp and polyGlu; amino acid sequencesderived from bone-targeting domains of bone proteins such as, e.g.,osteopontin, osteocalcin, and sialoprotein (Wang et al., Adv. DrugDelivery Rev., 57: 1049-76 (2005)); polymeric and non-polymericmolecules that reduce renal clearance such as, e.g., negatively chargedPEGs; and natural polymers (e.g., those containing amino acids, fattyacids and/or carbohydrates) and synthetic polymers (e.g., PEGs) thatincrease resistance of the CNP variant to NEP degradation by increasingthe total mass of the CNP variant to the ranges described generallyherein, e.g., from about 2.6 or 2.8 kDa to about 6 or 7 kDa;

(b) and (c) may be the wild-type Cys6 and Phe7, another natural aminoacid or an unnatural amino acid, or may contain a peptide bond isostereas described herein to increase resistance to NEP cleavage; and

(d) may be the wild-type Gly8, or may be a larger natural or unnatural(e.g., t-Bu-Gly) amino acid or peptidomimetic to reduce binding to NEP.

In one embodiment, such CNP variants contain at least one modified aminoacid at (b), (c) and/or (d).

Other peptide bonds within CNP may be cleaved even if CNP22 or a variantthereof has an NEP-resistant peptide bond or peptide bond isostere atCys6-Phe7, including the Gly8-Leu9, Lys10-Leu11, Arg13-Ile14,Ser16-Met17, and Gly19-Leu20 bonds. Therefore, the disclosureencompasses CNP variants having peptide bond isostere(s) at one or moreother NEP cleavages sites in addition to the Cys6-Phe7 bond, wherein thepeptide bond isosteres include those described herein.

In another embodiment, the disclosure encompasses CNP variants having acysteine analog at Cys6 and/or Cys22, including but not limited tohomocysteine, penicillamine, 2-mercaptopropionic acid, and3-mercaptopropionic acid. In an embodiment, such CNP variants have acyclic domain formed by a disulfide bond between the wild-type Cys6 oranalog and Cys22 or analog.

In yet another embodiment, one or more residues of CNP22 or a variantthereof, up to all the residues, are substituted with a D-amino acid.Substitution of an L-amino acid with a D-amino acid essentially movesthe side chain about 120 degrees from its original position, therebypotentially disrupting the binding of the CNP peptide to NEP. In aspecific embodiment, L-Phe at Phe7 is substituted with its D-enantiomer,D-Phe.

In still another embodiment, a beta amino acid such as, e.g.,3-amino-2-phenylpropionic acid (or 2-phenyl-beta-alanine), replaces thewild-type alpha-amino acid Phe7. Use of a beta-amino acid effectivelyincreases the peptide backbone length by one methylene unit. Proteaseresistance can result from the change in substrate conformation or theincreased distance between amino acid side chains.

Non-limiting examples of variants of CNP22 having an unnaturalalpha-amino acid, a beta-amino acid or a peptide bond isostere include:

(SEQ ID NO: 56) GLSKGC(CH₂NH)FGLKLDRIGSMSGLGC (Analog A),(SEQ ID NO: 57) GLSKGC-(N-Me-Phe)-GLKLDRIGSMSGLGC (Analog B),(SEQ ID NO: 136) GLSKGC-(D-Phe)-GLKLDRIGSMSGLGC (Analog E),(SEQ ID NO: 58) GLSKGCF-(tBu-Gly)-LKLDRIGSMSGLGC (Analog F),(SEQ ID NO: 137) GLSKGC-(3-Cl-Phe)-GLKLDRIGSMSGLGC (Analog G), and(SEQ ID NO: 59) GLSKGC-[NHCH₂CH(Ph)CO]-GLKLDRIGSMSGLGC (Analog H,formed using 3-amino-2-phenylpropionic acid).

In a further embodiment, the CNP variants have a total masscharacterized by the ranges described generally herein, e.g., from about2.6 or 2.8 kDa to about 6 or 7 kDa, designed for increased resistance toNEP degradation, and are represented by the formula:

-   (x)-Gly₁-Leu₂-Ser₃-(a)₄-Gly₅-(b)₆-(c)₇-(d)₈-(e)₉-(f)₁₀-(g)₁₁-Asp₁₂-Arg₁₃-(h)₁₄-Gly₁₅-Ser₁₆-(i)₁₇-Ser₁₈-Gly₁₉-(j)₂₀-Gly₂₁-Cys₂₂-(z)    (SEQ ID NO: 46), wherein:

(x) and (z) independently may be absent or may be selected from thegroup consisting of synthetic bone-targeting compounds such as, e.g.,bisphosphonates; amino acid sequences useful in bone or cartilagetargeting such as, e.g., polyAsp and polyGlu; amino acid sequencesderived from bone-targeting domains of bone proteins such as, e.g.,osteopontin, osteocalcin, and sialoprotein; polymeric and non-polymericmoieties that reduce renal clearance such as, e.g., negatively chargedPEGs; polymers containing, e.g., amino acids, hydrophobic acids, and/orcarbohydrates; and synthetic hydrophilic polymers such as, e.g., PEGs;

(a) may be the wild-type Lys at that position or may be replaced with aconservative amino acid substitution or a natural or unnatural aminoacid or peptidomimetic that does not have a reactive primary amine on aside chain, including but not limited to Arg, Gly, 6-hydroxy-norleucine,citrulline (Cit), Gln, Ser or Glu, wherein in one embodiment (a) is Arg;

(b) is selected from the group consisting of Cys and peptide-bondisosteres between Cys6 and Phe7 such as, e.g., Cys-CH₂—NH;

(c) is selected from the group consisting of L-Phe; D-Phe;3-amino-2-phenylpropionic acid; N-alkylated derivatives of Phe, whereinthe N-alkyl group is methyl, ethyl, n-propyl, isopropyl, cyclopropyl,n-butyl, isobutyl, sec-butyl or tert-butyl; and Phe analogs, wherein oneor more ortho-, meta-, and/or para-positions of the benzene ring of thePhe analog are substituted with one or more substituents selected fromthe group consisting of halogen, hydroxyl, cyano, straight or branchedC₁₋₆ alkyl, straight or branched C₁₋₆ alkoxy, straight or branchedhalo-C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl, heterocyclyl, C₆₋₁₄ aryl andheteroaryl (examples include, but are not limited to, tyrosine,3-chlorophenylalanine, 2,3-chloro-phenylalanine,3-chloro-5-fluoro-phenylalanine,2-chloro-6-fluoro-3-methyl-phenylalanine), or wherein the benzene ringof the Phe analog can be replaced with another aryl group (non-limitingexamples include 1- and 2-naphthylalanine) or with a heteroaryl group(non-limiting examples include pyridylalanine, thienylalanine andfurylalanine);

(d) is selected from the group consisting of Gly, tert-butyl-Gly(tBu-Gly), Thr, Ser, Val and Asn;

(e) is selected from the group consisting of Leu, Ser, Thr andpeptide-bond isosteres such as, e.g., N-Me-Leu;

(f) may be the wild type Lys at that position or may be replaced with aconservative amino acid substitution or a natural or unnatural aminoacid or peptidomimetic that does not have a reactive primary amine on aside chain, including but not limited to Arg, Gly, 6-hydroxy-norleucine,citrulline (Cit), Gln, Ser or Glu, wherein in one embodiment (f) is notArg;

(g) is selected from the group consisting of Leu and peptide-bondisosteres such as, e.g., N-Me-Leu;

(h) is selected from the group consisting of Ile, tBu-Gly, andpeptide-bond isosteres such as, e.g., N-Me-Ile;

(i) is selected from the group consisting of Met, Val, Asn, beta-Cl-Ala,2-aminobutyric acid (Abu) and 2-amino-isobutyric acid (Aib); and

(j) is selected from the group consisting of Leu, norleucine (Nle),homoleucine (Hleu), Val, tert-butyl-Ala (tBu-Ala), Ser, Thr, Arg, andpeptide-bond isosteres such as, e.g., N-Me-Leu.

In another embodiment, the CNP variants have a total mass characterizedby the ranges described generally herein, e.g., from about 2.6 or 2.8kDa to about 6 or 7 kDa, designed for increased resistance to NEPcleavage, and are represented by the formula:

-   (x)-Gly₁-Leu₂-Ser₃-(a)₄-Gly₅-(b)₆-(c)₇-(d)₈-(e)₉-(f)₁₀-(g)₁₁-Asp₁₂-Arg₁₃-(h)₁₄-(i)₁₅-Ser₁₆-(j)₁₇-Ser₁₈-Gly₁₉-(k)₂₀-Gly₂₁-Cys₂₂-(z)    (SEQ ID NO:143), wherein:

(x) and (z) independently may be absent or may be selected from thegroup consisting of synthetic bone-targeting compounds such as, e.g.,bisphosphonates; amino acid sequences useful in bone or cartilagetargeting such as, e.g., polyAsp and polyGlu; amino acid sequencesderived from bone-targeting domains of bone proteins and derivativesthereof, such as, e.g., fusion proteins or peptide sequences ofosteopontin, osteocalcin, sialoprotein, etc.; moieties that reduce renalclearance, including but not limited to hydrophilic or water-solublepolymers such as, e.g., charged PEG molecules; and moieities comprising,e.g., PEGs, amino acids, carbohydrates, and/or hydrophobic acids;

(a) may be the wild type Lys at that position or may be replaced with aconservative amino acid substitution or a natural or unnatural aminoacid or peptidomimetic that does not have a reactive primary amine on aside chain, including but not limited to Arg, Gly, 6-hydroxy-norleucine,citrulline (Cit), Gln, Ser or Glu, wherein in one embodiment (a) is Arg;

(b) is selected from the group consisting of Cys and peptide bondisosteres between Cys6 and Phe7 such as, e.g., Cys-CH₂—NH;

(c) is selected from the group consisting of L-Phe; D-Phe;3-amino-2-phenylpropionic acid; N-alkylated derivatives of Phe, whereinthe N-alkyl group is methyl, ethyl, n-propyl, isopropyl, cyclopropyl,n-butyl, isobutyl, sec-butyl or tert-butyl; and Phe analogs, wherein oneor more ortho-, meta-, and/or para-positions of the benzene ring of thePhe analog are substituted with one or more substituents selected fromthe group consisting of halogen, hydroxyl, cyano, straight or branchedC₁₋₆ alkyl, straight or branched C₁₋₆ alkoxy, straight or branchedhalo-C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₄ aryl, heterocyclyl andheteroaryl (examples include, but are not limited to, tyrosine,3-chlorophenylalanine, 2,3-chloro-phenylalanine,3-chloro-5-fluoro-phenylalanine,2-chloro-6-fluoro-3-methyl-phenylalanine), or wherein the benzene ringof the Phe analog can be replaced with another aryl group (non-limitingexamples include 1- and 2-naphthylalanine) or with a heteroaryl group(non-limiting examples include pyridylalanine, thienylalanine andfurylalanine);

(d) is selected from the group consisting of Gly, tert-butyl-Gly, Thr,Ser, Val and Asn;

(e) is selected from the group consisting of Leu, Ser, Thr, and peptidebond isosteres such as, e.g., N-Me-Leu;

(f) is selected from the group consisting of Lys, Arg, Gly,6-hydroxy-norleucine, citrulline (Cit), Gln and Ser;

(g) is selected from the group consisting of Leu, Asn, and peptide bondisosteres such as, e.g., N-Me-Leu;

(h) is selected from the group consisting of Ile, tert-butyl-Gly(tBu-Gly), Asn, and peptide bond isosteres such as, e.g., N-Me-Ile;

(i) is selected from the group consisting of Gly, Arg, Ser and Asn;

(j) is selected from the group consisting of Met, Val, Asn, beta-Cl-Ala,2-aminobutyric acid (Abu) and 2-amino-isobutyric acid (Aib); and

(k) is selected from the group consisting of Leu, norleucine (Nle),homoleucine (Hleu), Val, tert-butyl-Ala (tBu-Ala), Arg, Thr, Ser, andpeptide bond isosteres such as, e.g., N-Me-Leu.

To improve the delivery of the CNP variants to the target sites ofbone-related disorders (e.g., skeletal dysplasias), the CNP variants canbe attached (e.g., at the N-terminus and/or C-terminus) to bone- orcartilage-targeting moieties. Non-limiting examples of bone- orcartilage-targeting moieties include bisphosphonates; hydroxyapatite;glucosamine; collagen (e.g., collagen type X); polyAsp; polyGlu; andamino acid sequences derived from bone-targeting domains of boneproteins such as, e.g., osteocrin, osteopontin, osteocalcin, andsialoprotein.

In addition to being less susceptible to NEP cleavage, the CNP variantspotentially have reduced affinity to the NPR-C clearance receptor, whileretaining CNP functionality. Besides NEP-mediated degradation, thehalf-life of CNP22 is influenced by the clearance receptor, NPR-C, whichshares 58% sequence homology with the extracellular peptide-bindingdomain of NPR-B. CNP22 binds tightly to not only NPR-B (7-30 pMaffinity), but also NPR-C (11-140 pM) (Bennett, B. D. et al., J. Biol.Chem., 266: 23060-67 (1991); Koller K. J. & Goeddel, D. V., Circulation,86: 1081-88 (1992); Suga, S. et al., Endocrinology, 130: 229-39 (1992)).Even though the NPR-B crystal structure has yet to be reported, sequencehomology as well as similarities between the NPR-C and NPR-A crystalstructures (He, X.-L. et al., Science, 293(5535): 1657-62 (2001); Ogawa,H. et al., J. Biol. Chem., 279(27): 28625-31 (2004); He, X.-L., J. Mol.Biol., 361(4): 698-714 (2006)) suggest that NPR-B likely assumes asimilar overall structural fold.

Therefore, an NPR-B homology model was built based on structure-basedsequence alignment and crystallographic structures of the followingrelated systems: CNP bound to NPR-C, ANP bound to NPR-A, and ANP boundto NPR-C (He, X.-L. et al., Science, 293(5535): 1657-62 (2001); Ogawa,H. et al., J. Biol. Chem., 279(27): 28625-31 (2004); He, X.-L., J. Mol.Biol., 361(4): 698-714 (2006)). Based on observations that the receptorappears to determine the bound peptide conformation, and that NPR-B mostclosely resembles NPR-A with respect to primary structure and functionalproperties, the NPR-B/CNP homology model was built with the NPR-A/ANPcrystal structure as a model. Published signaling data of CNP variants(U.S. Pat. No. 5,434,133 and US Patent Application Publication No.2004/0138134 A1), and of functional ANP variants that no longer bind toNPR-C (Cunningham, EMBO 13(11) 2508-15, 1994) were used to refine andinterpret the NPR-B/CNP model.

The present disclosure encompasses CNP variants designed for improvedNPR-B selectivity based on a homology-based structural model of theNPR-B/CNP complex. Combining the experimental and computationalstructure data of natriuretic peptides bound to the various receptorswith the published functional data, CNP variants were generated thatcontinue to bind to NPR-B, but can potentially have reduced affinity tothe NPR-C clearance receptor. For example, NPR-C has a unique insertionin a loop structure in the peptide-binding site, placing its loopresidues closer to such peptide residues as CNP Gly8 (or ANP Gly9),compared to respective loop residues in NPR-A and NPR-B. Earlier studiesindicated that the G9T mutation in ANP contributes to reduce affinity toNPR-C, thereby improving NPR-A selectivity (Cunningham, EMBO J., 13(11):2508-15 (1994)). Accordingly, CNP variants were generated to replace thecorresponding Gly8 residue with a larger residue (Ser, Val, Thr or Asn)to disrupt the CNP binding to NPR-C without affecting its binding toNPR-B. Further, one or more mutations were introduced at the C-terminalend of CNP, encompassing Gly15 to Gly21, which is predicted to interactwith receptor-specific residues, based on the detailed structuralanalyses of the receptor/peptide complexes. For example, a G19R mutationin CNP22 does not result in a significant loss of NPR-B signalingactivity. This mutation, however, cannot be modeled into the availablecrystal structure of NPR-C/CNP without altering the conformations ofneighboring residues. These observations suggest that the G19R mutationmay selectively disrupt the binding of CNP to a particular receptor,such as NPR-C.

In an embodiment, the CNP variants have substitution(s) at one or moreGly sites at positions 1, 5, 8, 15, 19 and 21, to reduce conformationalflexibility and thereby increase receptor specificity. Comparativeanalyses of crystal structures of ANP bound to NPR-C and NPR-A (Ogawa,H. et al., J. Biol. Chem., 279: 28625-31 (2004); He, X.-L., J. Mol.Biol., 361: 698-714 (2006)) indicate that the conformational flexibilityof ANP may play an important role in determining the receptorselectivity.

In one embodiment, functional CNP variants with potentially reducedaffinity to NPR-C have one or more of the following amino acidsubstitutions: G1R, G1E, G5R, G5Q, G5S, F7Y, G8T, G8S, G8V, G8N, L9S,L9T, K10Cit, K10Q, K10S, 114N, G15R, G15S, G15N, G15Cit, S16Q, M17V,M17N, G19S, G19R, G19N, L20V, L20R, L20T, L20S, G21S, G21T and G21R. Inan embodiment, the CNP variants have multipoint substitutions atpositions 1, 5, 7, 8, 9, 10, 14, 15, 16, 17, 19, 20 and/or 21, and mayoptionally have modifications at any of the other positions in thepeptide sequence of the variant.

In a further embodiment, the CNP variants described herein may beconjugated to moieties, up to a total mass characterized by the rangesdescribed generally herein, e.g., from about 2.6 or 2.8 kDa to about 6or 7 kDa, at the N-terminus, the C-terminus and/or an internal site, tofacilitate bone/cartilage targeting, reduce NPR-C and renal clearance,increase resistance to NEP degradation, and/or improve CNPfunctionality. In one embodiment, the CNP variants are not conjugated toa polymeric moiety at a site within the cyclic region (corresponding toCys6 to Cys22 of CNP22). Non-limiting examples of polymeric ornon-polymeric moieties that can be conjugated to the CNP variantsinclude synthetic bone-targeting compounds such as, e.g.,bisphosphonates; bone/cartilage targeting peptide sequences such as,e.g., polyAsp and polyGlu; peptide sequences derived from bone-targetingdomains of bone proteins such as, e.g., osteopontin, osteocalcin andsialoprotein; peptide sequences derived from the functional domains ofbone morphogenetic proteins such as, e.g., BMP2, BMP3, BMP5, BMP7 andBMP8a; peptide sequences derived from polypeptides of natriuretic originsuch as, e.g., NPPC, ANP and BNP; other natural polymeric ornon-polymeric moieties such as, e.g., carbohydrates, fatty acids andphospholipids; biocompatible synthetic hydrophilic polymers such as,e.g., PEG (or PEO); hydrophobic polymeric or non-polymeric moieties suchas, e.g., heptanoic acid and pentanoic acid; and combinations thereof.

The CNP variants described herein can have substantially similar orbetter functional activity than CNP22, e.g., with respect to stimulationof cGMP production and signaling. In one embodiment, the CNP variants invitro or in vivo stimulate the production of at least about 50% of thecGMP level produced under the same concentration of wtCNP22 (e.g., 1uM). In certain embodiments, the CNP variants retain at least about 50%,60%, 70%, 80%, 90%, 95% or 100% of the cGMP-stimulation activity ofwild-type CNP22 in vitro or in vivo. In another embodiment, the CNPvariants have improved cGMP-stimulation activity compared to CNP22. Incertain embodiments, the CNP variants in vitro or in vivo stimulate theproduction of at least about 110%, 120%, 130%, 140%, 150%, 200%, 250%,300%, 350%, 400%, 450%, 500% or more of the cGMP level produced underthe same concentration of wtCNP22 (e.g., 1 uM).

Optionally excluded from the present disclosure are any of thenatriuretic (e.g., CNP) peptides, fragments and variants specificallydisclosed, and any of the natriuretic (e.g., CNP) peptides, fragmentsand variants actually produced, in any of the prior publicationsreferenced herein, including but not limited to, U.S. Pat. Nos.5,434,133, 6,034,231, 6,020,168, 6,743,425, 7,276,481, WO 94/20534, WO02/047871, WO 2005/098490, WO 2004/047871, EP 0497368, EP 0466174, andFuruya et al., Biochem. Biophys. Res. Comm. 183: 964-969 (1992)). Allsuch documents are incorporated by reference herein in their entirety.

In one embodiment, the present disclosure optionally excludes all knownwild-type CNP-53, wild-type CNP-22, wild-type CNP-17, wild-type BNP, andwild-type ANP of human origin and non-human origin. For example, in anembodiment the disclosure optionally excludes human CNP-17, humanCNP-22, chicken CNP-22 (corresponding to hCNP-22(Leu9Val)), trout andeel CNP-22 (corresponding to hCNP-22(Leu2Trp, Ser3Asn, Lys4Arg)), frogCNP22-I (corresponding to hCNP-22(Leu2Tyr, Lys4Arg, Leu9Val, Ser16Ala,Met17Phe)), frog CNP22-II (corresponding to hCNP-22(Leu2Thr, Ser16Ala)),human CNP-53, and pig and rat CNP-53 (corresponding to hCNP-53(Gln17His,Ala28Gly)). In another embodiment, the disclosure optionally excludesfragments of NPPC, proCNP and CNP-53 which are produced by proteolyticcleavage in vivo in humans and non-human animals. In yet anotherembodiment, optionally excluded from the disclosure are the followingtruncated fragments of wild-type human CNP-53: CNP-50, CNP-46, CNP-44,CNP-39, CNP-30, CNP-29, CNP-28, CNP-27 and CNP-26.

In a further embodiment, the present disclosure optionally excludes CNPpeptides and fragments thereof isolated or sought from the shark speciesTriakis scyllia and Scyliorhinus canicula (see, e.g., M. Takano et al.,Zool. Sci., 11: 451-454 (1994)), including:

(SEQ ID NO: 204) RLLKDLSNNPLRFRGRSKKGPSRGCFGVKLDRIGAMSGLGC (CNP-41);(SEQ ID NO: 205) LKDLSNNPLRFRGRSKKGPSRGCFGVKLDRIGAMSGLGC (CNP-39);(SEQ ID NO: 206) KDLSNNPLRFRGRSKKGPSRGCFGVKLDRIGAMSGLGC (CNP-38); and(SEQ ID NO: 207) GPSRGCFGVKLDRIGAMSGLGC (CNP-22).

In another embodiment, optionally excluded from the disclosure are CNPpeptides and fragments thereof isolated or sought from the shark speciesLamna ditropis (see, e.g., M. Takano et al., Zool. Sci., 11: 451-454(1994)), including:

(SEQ ID NO: 208) RLLKDLSNNPLRFKGRSKKGPSRGCFGVKLDRIGAMSGLGC (CNP-41);(SEQ ID NO: 209) LKDLSNNPLRFKGRSKKGPSRGCFGVKLDRIGAMSGLGC (CNP-39);(SEQ ID NO: 210) KDLSNNPLRFKGRSKKGPSRGCFGVKLDRIGAMSGLGC (CNP-38);(SEQ ID NO: 211) FKGRSKKGPSRGCFGVKLDRIGAMSGLGC (CNP-29); and(SEQ ID NO: 212) GPSRGCFGVKLDRIGAMSGLGC (CNP-22).

In still another embodiment, optionally excluded from the disclosure areCNP peptides and fragments thereof isolated from the shark speciesSqualus acanthias (see, e.g., M. Takano et al., Zool. Sci., 11: 451-454(1994)), including:

(SEQ ID NO: 213) RLLQDLSNNPLRFKGRSKKGPSRSCFGLKLDRIGAMSGLGC (CNP-41); and(SEQ ID NO: 214) GPSRSCFGLKLDRIGAMSGLGC (CNP-22).

In a further embodiment, the present disclosure optionally excludes CNPpeptides isolated from medaka and puffer fish, designated “CNP-1”,“CNP-2”, “CNP-3” and “CNP-4” in K. Inoue et al., Proc. Nat. Acad. Sci.,100(17): 10079-10084 (2003):

(SEQ ID NO: 215) GWNRGCFGLKLDRIGSMSGLGC (medaka and puffer fish CNP-1);(SEQ ID NO: 216) PMVAGGGCFGMKMDRIGSISGLGC (medaka CNP-2);(SEQ ID NO: 217) GRSSMVGGRGCFGMKIDRIGSISGLGC (puffer fish CNP-2);(SEQ ID NO: 218) GGMRSCFGVRLERIGSFSGLGC (medaka CNP-3); (SEQ ID NO: 219)GGLRSCFGVRLARIGSFSGLGC (puffer fish CNP-3); (SEQ ID NO: 220)GGSTSRSGCFGHKMDRIGTISGMGC (medaka CNP-4); and (SEQ ID NO: 221)GGSSRSGCFGHKMDRIGTISGMGC (puffer fish CNP-4).

In a still further embodiment, optionally excluded from the disclosureare CNP-39 isolated from platypus venom and the CNP-22 fragment thereof,designated “ovCNP-39” and “ovCNP-39(18-39)” in G. de Plater et al.,Toxicon., 36(6): 847-857 (1998):

(SEQ ID NO: 222) LLHDHPNPRKYKPANKKGLSKGCFGLKLDRIGSTSGLGC (ovCNP-39); and(SEQ ID NO: 223) GLSKGCFGLKLDRIGSTSGLGC (ovCNP-39(18-39).

In another embodiment, the present disclosure optionally excludes thefollowing peptides as specifically disclosed in US 2007/0197434:

(SEQ ID NO: 224) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ala-Met-Ser-Gly-Leu-Gly-Cys; (SEQ ID NO: 225)Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Gln-Ser-Gly-Leu-Gly-Cys; (SEQ ID NO: 226)Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Ala-Ser-Gly-Leu-Gly-Cys; (SEQ ID NO: 227)Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys; (SEQ ID NO: 228)Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys-Asn-Ser- Phe-Arg-Tyr; and(SEQ ID NO: 229) Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr.In yet another embodiment, the disclosure optionally excludes peptidesof SEQ ID NO:10, disclosed generically in US 2007/0197434, wherein suchpeptides are CNP-17 variants having certain natural amino acidsubstitution(s) at position(s) 4, 5, 6, 11, 12, 14 and/or 15. In stillanother embodiment, optionally excluded from the disclosure are peptidescorresponding to hCNP-53(Ser47Ala), hCNP-53(Met48Gln),hCNP-53(Met48Ala), and hCNP-53(C-term.)-Asn-Ser-Phe-Arg-Tyr.

In an embodiment, the present disclosure optionally excludes thepeptides of SEQ ID NOs 1-4 and 6-71 as specifically disclosed in U.S.Pat. No. 7,276,481. In another embodiment, the disclosure optionallyexcludes peptides of SEQ ID NO 5, disclosed generically in U.S. Pat. No.7,276,481, wherein such peptides are variants of CNP17 having at leastone natural amino acid substitution at Leu9, Lys10, Leu11, Ser16, Met17,Gly19, and/or Leu20. In still another embodiment, optionally excludedare CNP17 variants in which CNP17 or variants thereof contain N-Me-Phe7,or N-Me-Phe7 and N-Me-Leu11. In a further embodiment, the disclosureoptionally excludes CNP17 variants of SEQ ID NO 5, as disclosed in U.S.Pat. No. 7,276,481, which are fused or conjugated to growth hormone(GH), insulin-like growth factor 1 (IGF-1), or thyroid hormone (TH). Inyet another embodiment, optionally excluded are CNP22 variants in whichCNP22 is fused to GH, IGF-1 or TH, or attached to GH, IGF-1 or TH via alinker (e.g., a peptide linker). In still another embodiment, optionallyexcluded are CNP17 variants in which CNP17 or variants thereof areconjugated to biotin or fluorescein at the N-terminus or the C-terminus.

In a further embodiment, the present disclosure optionally excludes thepeptides of Compound Nos. 1-27, and SEQ ID NOs 1-17, 22-24, 30, 31 and40-42 as specifically disclosed in U.S. Pat. No. 5,434,133. In anotherembodiment, the disclosure optionally excludes peptides of SEQ ID NOs18-21 and 25-29, disclosed generically in U.S. Pat. No. 5,434,133. Instill another embodiment, the disclosure optionally excludes thepeptides of SEQ ID NOs 1-4 and 9 as specifically disclosed in WO94/20534.

In some embodiments, however, the disclosure still encompasses methodsof use of the natriuretic (e.g., CNP) peptides, fragments and variantsoptionally excluded herein, as well as pharmaceutical compositions(including sterile pharmaceutical compositions) comprising suchnatriuretic (e.g., CNP) peptides, fragments and variants.

C. Synthesis and Purification of CNP Variants

In some embodiments, the CNP variants described herein are produced byrecombinant expression, using certain techniques known in the art incertain embodiments. See, e.g., Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual, Second Edition. Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. (1989)); DNA Cloning: APractical Approach, Volumes I and II, D. N. Glover, Ed. (1985); andCurrent Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

In certain embodiments, the CNP variants described herein are producedby a recombinant process that comprises culturing in a medium a hostcell comprising a first polynucleotide encoding a CNP variantpolypeptide linked to a second polynucleotide encoding a cleavablepeptide or protein under conditions that result in expression of afusion polypeptide encoded by the polynucleotides, wherein the fusionpolypeptide comprises the CNP variant polypeptide directly linked to thecleavable peptide or protein or indirectly linked thereto via a linker.In some embodiments, the host cell is transformed with an expressionvector comprising the polynucleotide encoding the CNP variantpolypeptide linked to the polynucleotide encoding the cleavable peptideor protein. In certain embodiments, the fusion polypeptide is expressedas a soluble protein or as an inclusion body. The expressed fusionpolypeptide can be isolated from the host cell or culture medium, andthe isolated fusion polypeptide can be contacted with a cleaving agentto release the CNP variant.

Host cells used to produce CNP variants can be bacterial, yeast, insect,non-mammalian vertebrate, or mammalian cells. Bacterial cells includewithout limitation E. coli cell lines and strains. Non-limiting examplesof E. coli cell lines and strains include BL21, BL21(DE3),BL21(DE3)pLysS, BL21(DE3)pGro7, ArcticExpress(DE3), C41 [also calledC41(DE3)], C43 [also called C43(DE3)], Origami B(DE3), OrigamiB(DE3)pLysS, KRX, and Tuner(DE3). In an embodiment, CNP variants and CNPfusion proteins are produced using BL21(DE3) cells. Mammalian cellsinclude, but are not limited to, hamster, monkey, chimpanzee, dog, cat,bovine, porcine, mouse, rat, rabbit, sheep and human cells. The hostcells can be immortalized cells (a cell line) or non-immortalized(primary or secondary) cells and can be any of a wide variety of celltypes, such as, but not limited to, fibroblasts, keratinocytes,epithelial cells (e.g., mammary epithelial cells, intestinal epithelialcells), ovary cells (e.g., Chinese hamster ovary or CHO cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), chondrocytes and otherbone-derived cells, and precursors of these somatic cell types. Hostcells containing the CNP variant DNA or RNA are cultured underconditions appropriate for growth of the cells, expression of the DNA orRNA and identification/selection of cells expressing the CNP variant.

In some embodiments, the host cells are grown or cultured for a timeperiod at a temperature from about 10° C. to about 40° C., or from about20° C. to about 40° C., or from about 30° C. to about 40° C. In certainembodiments, the host cells are grown or cultured for a time period atabout 20° C., 22° C., 25° C., 28° C., 30° C., 35° C. or 37° C. Incertain embodiments, the host cells are grown or cultured for a timeperiod at about 35° C. or 37° C.

Recombinant polynucleotides encoding CNP variant polypeptides (includingCNP fusion proteins) are expressed in an expression vector comprising arecombinant polynucleotide comprising expression control sequencesoperatively linked to a nucleotide sequence to be expressed. Anexpression vector comprises sufficient cis-acting elements forexpression; other elements for expression can be supplied by the hostcell or in vitro expression system. Expression vectors include all thoseknown in the art, including without limitation cosmids, plasmids (e.g.,naked or contained in liposomes) and viruses that incorporate therecombinant polynucleotide. The expression vector is inserted into anappropriate host cell, via transformation or transfection, forexpression of the polynucleotide encoding the polypeptide (see, e.g.,Sambrook et al. (supra)).

Non-limiting examples of expression vectors contemplated for productionof CNP variants, including cleavable CNP fusion proteins, includepJexpress, pJexpress401, pJexpress404, pET-15b, pET-21a, pET-22b,pET-31b, pET-32a, pET-41a, pMAL, pMAL-c2X, pQE-30, pET-SUMO, and pTYB11.Expression of particular constructs can generate soluble CNP variants(including CNP fusion proteins) or insoluble CNP variants (including CNPfusion proteins) in the form of inclusion bodies.

In some embodiments, expression of the polynucleotide(s) encoding a CNPvariant or CNP fusion protein is enhanced using an isopropylβ-D-1-thiogalactopyranoside (IPTG)-inducible vector. In someembodiments, the host cells are grown or cultured for a time period at atemperature from about 10° C. to about 40° C., or from about 20° C. toabout 40° C., or from about 30° C. to about 40° C., in the presence ofIPTG. In certain embodiments, the host cells are grown or cultured for atime period at about 20° C., 22° C., 25° C., 28° C., 30° C., 35° C. or37° C. in the presence of IPTG. In certain embodiments, the host cellsare grown or cultured for a time period at about 35° C. or 37° C. in thepresence of 1 mM IPTG.

In further embodiments, the host cells are cultured with IPTG at aconcentration from about 0.4 mM to about 2 mM, or from about 0.4 mM toabout 1.5 mM, or from about 0.4 mM to about 1 mM. In certainembodiments, the IPTG is at a concentration of about 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mM. In anembodiment, the concentration of IPTG is about 1 mM.

In certain embodiments, the CNP variants described herein arerecombinantly expressed as fusion proteins comprising a CNP variantpolypeptide and a cleavable carrier protein or cleavable tag (e.g.,peptide tag), wherein the fusion protein comprises the CNP variantpolypeptide directly linked to the cleavable carrier protein or tag orindirectly linked thereto via a linker. Use of a carrier protein or tagfacilitates, e.g., detection, isolation and/or purification of thefusion protein. Cleavable carrier proteins and tags include, but are notlimited to, histidine (e.g., hexa-His) tags; human transcription factorTAF12 (TAF12), TAF12 fragments, TAF12 histone fold domain, mutants ofTAF12 and fragments thereof, TAF12(C/A), TAF12(D/E), TAF12(4D/4E),TAF12(6D/6E), TAF12(10D/10E), TAF12(C/A & D/E), TAF12(C/A & 4D/4E),TAF12(C/A & 6D/6E), TAF12(C/A & 10D/10E); ketosteroid isomerase (KSI);maltose-binding protein (MBP); β-galactosidase (β-Gal);glutathione-S-transferase (GST); thioredoxin (Trx); chitin bindingdomain (CBD); BMP-2, BMP-2 mutants, BMP-2(C/A); SUMO; and mutants andfragments thereof.

An expression construct can express a fusion protein comprising a CNPvariant and a carrier protein or tag. The tag can be an amino acidsequence that confers a useful property to the fusion protein. In oneembodiment, the tag is a ligand-binding domain that can be used topurify the fusion protein by applying the fusion protein to separationmedia containing the ligand. For example, a fusion protein comprising aglutathione-S-transferase (GST) domain can be applied to achromatographic column containing glutathione-linked separation media.As another example, a fusion protein comprising maltose-binding protein(MBP) as a tag can be applied to separation media containing maltose. Asa further example, a fusion protein comprising a polyhistidine tag maybe applied to a nickel column, whereby chelation of the polyhistidinetag to the nickel column facilitates purification of the fusion protein.In another embodiment, the tag is a ligand. For example, a fusionprotein can comprise glutathione as a tag and be applied to achromatographic column containing glutathione-S-transferase-linkedseparation media. Non-limiting examples of carrier proteins and tags foruse in fusion proteins include human transcription factor TAF12 (TAF12),ketosteroid isomerase (KSI), maltose-binding protein (MBP),β-galactosidase (β-Gal), glutathione-S-transferase (GST), thioredoxin(Trx), chitin-binding domain (CBD), BMP-2 mutation (BMPM), SUMO, CAT,TrpE, staphylococcal protein A, streptococcal proteins, starch-bindingprotein, cellulose-binding domain of endoglucanase A, cellulose-bindingdomain of exoglucanase Cex, biotin-binding domain, recA, Flag,poly(His), poly(Arg), poly(Asp), poly(Gln), poly(Phe), poly(Cys), greenfluorescent protein, red fluorescent protein, yellow fluorescentprotein, cyan fluorescent protein, biotin, avidin, streptavidin,antibody epitopes, and mutants and fragments thereof.

To generate the target CNP variant, the carrier protein or tag can becleaved from the fusion protein by means of chemical cleavage, proteasecleavage, or protein self-cleavage. Exemplary chemical and proteolyticcleavage agents (cleavage sites in parenthesis) include, but are notlimited to, formic acid (Asp-Pro), cyanogen bromide (CNBr) (Met-X),hydroxylamine (Asn-Gly), Factor Xa (IEGR-X) (SEQ ID NO: 230),Enterokinase (DDDDK-X) (SEQ ID NO: 231), ProTEV (EXXYXQ-G) (SEQ ID NO:232), and SUMO protease. Due to the nature of the particular kinds ofchemical cleavage, cleavage using formic acid may generate Pro-CNP, CNBrmay generate CNP having Met-to-Asn substitution, and hydroxylamine maygenerate Gly-CNP. Alternatively, chemical or protease cleavage may beavoided by using particular constructs (e.g., pET-21a-CNP) that expressCNP variants not as fusion proteins. Expression of pET-21a-CNP mayproduce Met-CNP. Or certain fusion proteins (e.g., those containingintein-CBD) can undergo self-cleavage to generate CNP.

In further embodiments, a fusion protein comprises a cleavable peptidelinker between a CNP variant and a carrier protein or tag (e.g., peptidetag). In certain embodiments, the cleavable peptide linker is selectedfrom the group consisting of Asp-Pro, Asn-Gly, Met-X, Val-Asp-Asp-Arg(SEQ ID NO: 233), Gly-Ser-Asp-Arg (SEQ ID NO: 234), Ile-Thr-Asp-Arg (SEQID NO: 235), Pro-Gly-Asp-Arg (SEQ ID NO: 236), Ile-Glu-Gly-Arg-X (SEQ IDNO: 230), Asp-Asp-Asp-Asp-Lys-X (SEQ ID NO: 231), Glu-X-X-Tyr-X-Gln-Gly(SEQ ID NO: 232), Ala-Phe-Leu-Gly-Pro-Gly-Asp-Arg (SEQ ID NO: 237), andMGSSHHHHHHSSGLVPRGSHTGDDDDKHMD (pET-15b linker) (SEQ ID NO: 95), where Xdenotes an amino acid. In some embodiments, the cleavable peptide linkeris cleaved by a cleaving agent selected from the group consisting ofpalladium, cyanogen bromide (CNBr), formic acid, hydroxylamine,clostripain, thrombin, chymotrypsin, trypsin, trypsin-like proteases,carboxypeptidase, enterokinase (enteropeptidase), Kex 2 protease, Omp Tprotease, Factor Xa protease, subtilisin, proTEV, SUMO protease, V8protease, HIV protease, rhinovirus protease, furilisin protease, IgAproteases, human Pace protease, collagenase, Nia protease, poliovirus2Apro protease, poliovirus 3C protease, genenase, furin, elastase,Proteinase K, pepsin, rennin (chymosin), microbial aspartic proteases,papain, calpain, chymopapain, ficin (ficain), bromelain (bromelase),cathespisin B, caspases, thermolysin, Endoprotease Arg-C, EndoproteaseGlu-C, Endoprotease Lys-C, kallikrein, and plasmin.

In certain embodiments, the cleavable carrier protein, tag (e.g.,peptide tag) or peptide linker is cleaved using formic acid to releasethe CNP variant from the fusion protein. In some embodiments, the formicacid is at a concentration from about 1% to about 20%, or from about 1%to about 15%, or from about 2% to about 15%, or from about 1% to about10%, or from about 2% to about 10%, or from about 1% to about 5%, orfrom about 2% to about 5%. In certain embodiments, the formic acid is ata concentration of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14% or 15%. In certain embodiments, the formic acid is at aconcentration of about 2%, 5% or 10%.

In further embodiments, cleavage of the CNP fusion protein in thepresence of formic acid is conducted at a temperature from about 20° C.to about 80° C., or from about 30° C. to about 75° C., or from about 40°C. to about 75° C., or from about 50° C. to about 75° C., or from about50° C. to about 70° C., or from about 55° C. to about 70° C., or fromabout 50° C. to about 60° C. In some embodiments, cleavage in thepresence of formic acid is conducted at about 20° C., 22° C., 25° C.,30° C., 35° C., 37° C., 40° C., 42° C., 45° C., 50° C., 55° C., 60° C.,65° C., 70° C., 75° C. or 80° C. In certain embodiments, cleavage in thepresence of formic acid is conducted at about 50° C., 55° C., 60° C.,65° C. or 70° C. In certain embodiments, cleavage in the presence offormic acid is conducted at about 55° C. or 70° C.

In additional embodiments, cleavage of the CNP fusion protein in thepresence of formic acid is performed for a time period from about 3 hrto about 48 hr, or from about 5 hr to about 48 hr, or from about 5 hr toabout 36 hr, or from about 5 hr to about 24 hr, or from about 5 hr toabout 18 hr, or from about 20 hr to about 24 hr, or for about 6 hr toabout 10 hr. In certain embodiments, cleavage in the presence of formicacid is performed for about 5 hr, 6 hr, 12 hr, 15 hr, 18 hr, 20 hr or 24hr.

In some embodiments, cleavage of the CNP fusion protein is conducted inthe presence of about 2%, 5% or 10% formic acid at about 55° C. forabout 20 hr to about 36 hr, or at about 60° C. for about 15 hr to about24 hr, or at about 65° C. for about 10 hr to about 21 hr, or at about70° C. for about 6 hr to about 18 hr. In certain embodiments, cleavageof the CNP fusion protein is conducted in the presence of about 2%formic acid at about 55° C. for about 20 hr to about 24 or 36 hr, or atabout 60° C. for about 15 hr to about 24 hr, or at about 65° C. forabout 10 hr to about 18 hr, or at about 70° C. for about 6 hr to about10 hr.

The present disclosure provides mild conditions for cleavage of CNPfusion proteins using formic acid to afford high yields of CNP variants.The conditions described herein for fusion protein cleavage using formicacid are also suitable for cleavage of fusion proteins comprisingpolypeptides or proteins other than CNP, where the fusion proteinscontain an Asp-Pro peptide bond.

In further embodiments, soluble CNP fusion proteins or CNP fusionprotein inclusion bodies are treated with a buffer and/or a detergentprior to chemical cleavage (e.g., using formic acid) or proteolyticcleavage of the CNP fusion protein. Non-limiting examples of buffersinclude B-PER II; diluted B-PER II (e.g., 1/20 dilution); B-PER; B-PERphosphate buffer; buffers containing Tris (e.g., 25 mM Tris, pH 7.5);buffers containing Tris and NaCl (e.g., 25 mM Tris, 150 mM NaCl, pH7.9); and PBS. In an embodiment, the buffer is B-PER II. Non-limitingexamples of detergents include octylsucrose, Triton X-100, Tween-20,NP-40, and CA-630. The detergent can be in a buffer (e.g., 1% detergentin 25 mM Tris buffer, pH 7.5). In certain embodiments, the detergent isTriton X-100 or CA-630.

It is understood that any of the methods and conditions described abovemay be used in combination with any of the other methods and conditionsdescribed above to generate a CNP variant disclosed herein.

In other embodiments, the CNP variants described herein are synthesizedusing a peptide synthesizer and purified according to methods known inthe art, e.g., according to the methods of Atherton and Sheppard, SolidPhase Peptide Synthesis: a Practical Approach, IRL Press (Oxford,England (1989)).

Peptides can be synthesized based on, e.g., the following peptidesequence of CNP: G¹LS(K or R)GC⁶F⁷G⁸L(K or R or Nle or6-OH-Nle)LDRIGSMSGLGC²².

Exemplary CNP variants include but are not limited to:

Analog A (GLSKGC(CH2NH)FGLKLDRIGSMSGLGC) (SEQID NO: 56) was made by converting the backbone “—C═O”group of C⁶ to a “—CH₂” group;Analog B (GLSKGC(N—Me—Phe)GLKLDRIGSMSGLGC) (SEQID NO: 57) was made by converting the backbone “—NH”group of F⁷ to an “—N—CH₃” group;Analog E (GLSKGC(D-Phe)GLKLDRIGSMSGLGC) (SEQID NO: 136) was made using D-Phe at F⁷;Analog F (GLSKGCF(tBu-Gly)LKLDRIGSMSGLGC) (SEQID NO: 58) was made using a tertiary-butyl-Gly at G⁸;Analog G (GLSKGC(3-Cl-Phe)GLKLDRIGSMSGLGC) (SEQID NO: 137) was made by adding a chloride atom toa meta position of the phenyl ring of F⁷(similar variants can be generated by makingortho, meta and/or para substitutions of thephenyl ring of Phe7 with Cl, F, Br, OH and/or CH₃); andAnalog H (GLSKGC[NHCH₂CH(Ph)CO]GLKLDRIGSMSGLGC)(SEQ ID NO: 59) was made using (±)-3-(amino)-2-phenylpropionic acid at F⁷.

Examples of CNP variants having, e.g., amino acid extensions,substitutions with natural or unnatural amino acids or peptide bondisosteres, and/or conjugations to polymers or hydrophobic moieties,include without limitation:

Analog J (SEQ ID NO: 91) C6—CH2—NH, N—Me-L9, N—Me-L20 Analog K(SEQ ID NO: 92) N—Me-L9, N—Me-L20 Analog L (SEQ ID NO: 93)N—Me-L9, N—Me-L11, N—Me-L20 Analog M (SEQ ID NO: 94) N—Me-L9, N—Me-L11Analog Z (SEQ ID NO: 95) K4R, F7Y Analog AA (SEQ ID NO: 96) K4R, G8VAnalog AB (SEQ ID NO: 97) K4R, G8S Analog AC (SEQ ID NO: 98) K4R, G8TAnalog AD (SEQ ID NO: 99) K4R, L9T Analog AE (SEQ ID NO: 100) K4R, G15RAnalog AF (SEQ ID NO: 101) K4R, G15Cit Analog AG (SEQ ID NO: 102)K4R, M17V Analog AH (SEQ ID NO: 35) K4R Analog AJ (SEQ ID NO: 103)K4R, L20V Analog AK (SEQ ID NO: 104) K4R, L20t-Bu-Ala Analog AT(SEQ ID NO: 105) G1E, K4E Analog AV (SEQ ID NO: 106)G1E, K4E - pentanoic acid (attached at the N-terminus) Analog AW(SEQ ID NO: 107) G1E, K4E - heptanoic acid (attached at the N-terminus)Analog AX (SEQ ID NO: 2) CNP17 (delta N-term) Analog AY (SEQ ID NO: 36)GANRR-CNP22(K4R) Analog AZ (SEQ ID NO: 41) R-CNP22(K4R) Analog BB(SEQ ID NO: 108) G1E - heptanoic acid (attached at the N-terminus)Analog BC (SEQ ID NO: 109) G1E - pentanoic acid (attached at theN-terminus) Analog BF (SEQ ID NO: 110) K4R, K10Cit Analog BG(SEQ ID NO: 111) K4R, K10Q Analog BH (SEQ ID NO: 112) K4R, K10RAnalog BJ (SEQ ID NO: 113) K4R, G15N Analog BK (SEQ ID NO: 114)K4R, G15S Analog BL (SEQ ID NO: 60) CNP-37 (SEQ ID NO: 4) CNP-53Analog CA (SEQ ID NO: 61) AAWARLLQEHPNA-CNP22 Analog CB (SEQ ID NO: 62)AAWARLLQEHPNAR-CNP22 Analog CC (SEQ ID NO: 63) DLRVDTKSRAAWAR-CNP22Analog CD (SEQ ID NO: 68) SPKMVQGSG-CNP17-KVLRRH (N- and C-terminalBNP tails) Analog CE (SEQ ID NO: 81) GERAFKAWAVARLSQ-CNP22 (HSA-CNP22)Analog CF (SEQ ID NO: 79) GQPREPQVYTLPPS-CNP22 PEG(24K)-CNP22PEG(20K)-CNP22 PEG(5K)-CNP22 PEG(2K)-CNP22 PEG(2K)-CNP17 (SEQ ID NO: 36)PEG(1K)-GANRR-CNP22(K4R) PEG(1K)-CNP22 PEO4-(PEO12)3(branched)-CNP22PEO12-CNP22 (SEQ ID NO: 36) PEO12-GANRR-CNP22(K4R) (SEQ ID NO: 36)PEO24-GANRR-CNP22(K4R); andSEQ ID NOs: 1 to 6 and 34 to 144, and variantsthereof that comprise up to 1, 2, 3, 4, or 5 further modifications.

In one embodiment, the CNP variants are cyclized via formation of adisulfide bond between Cys⁶ and Cys²². Cys⁶ can be a cysteine analogsuch as, e.g., homocysteine or penicillamine. In a further embodiment,the CNP variants can be cyclized by a covalent bond formed head-to-tail,side chain-to-side chain, side chain-to-head, or side chain-to-tail. Inan embodiment, the covalent bond is formed between an amino acid at ortoward the N-terminus and an amino acid at or toward the C-terminus ofthe peptide (referred to as “terminal” amino acids in this context). Inanother embodiment, the covalent bond is formed between the side chainsof the two terminal amino acids. In yet another embodiment, the covalentbond is formed between the side chain of one terminal amino acid and theterminal group of the other terminal amino acid, or between the terminalgroups of the two terminal amino acids.

Head-to-tail cyclization of the terminal amine to the terminal carboxylgroup can be carried out using a number of methods, e.g., usingp-nitrophenyl ester, 2,4,5-trichlorophenyl ester, pentafluorophenylester, the azide method, the mixed anhydride method, HATU, a carbodimide(e.g., DIC, EDC or DCC) with a catalyst such as HOBt, HONSu or HOAt, oron-resin cyclization.

In addition, the cyclic structure can be formed via a bridging groupinvolving the side chains of amino acid residues of the CNP variantand/or the terminal amino acid residues. A bridging group is a chemicalmoiety that allows cyclization of two portions of the peptide.Non-limiting examples of bridging groups include amides, thioethers,thioesters, disulfides, ureas, carbamates, sulfonamides, and the like. Avariety of methods are known in the art for incorporation of unitshaving such bridging groups. For example, a lactam bridge (i.e., acyclic amide) can be formed between the N-terminal amino group or anamino group on a side chain and the C-terminal carboxylic acid or acarboxyl group on a side chain, e.g., the side chain of lysine orornithine and the side chain of glutamic acid or aspartic acid. Athioester can be formed between the C-terminal carboxyl group or acarboxyl group on a side chain and the thiol group on the side chain ofcysteine or a cysteine analog.

Alternatively, a cross link can be formed by incorporating a lanthionine(thio-dialanine) residue to link alanine residues that are covalentlybonded together by a thioether bond. In another method, a cross-linkingagent, such as a dicarboxylic acid (e.g., suberic acid (octanedioicacid)), can link the functional groups of amino acid side chains, suchas free amino, hydroxyl, and thiol groups.

Enzyme-catalyzed cyclization can also be used. For example, it has beenreported that the thioesterase domain of tyrocidine synthetase can beused to cyclize a thioester precursor, a subtilisin mutant can beutilized to cyclize peptide glycolate phenylalanylamide esters, and theantibody ligase 16G3 can be employed to cyclize a p-nitrophenylester.For a review of peptide cyclization, see Davies, J. Peptide Sci., 9:471-501 (2003), incorporated herein by reference in its entirety.

In certain embodiments, the final cyclized product has a purity of atleast about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at leastabout 99%.

D. Chemically Modified CNP Variants

Chemical modification of CNP22 or variants thereof can potentiallyimpart advantageous properties to the modified CNP peptides, such asincreased stability and half-life and reduced immunogenicity (for ageneral discussion of chemical modification of therapeutic proteins, seePharmazie, 57(1): 5-29 (2002)). For example, attaching natural orsynthetic, polymeric or non-polymeric moieties (e.g., PEG) to CNPpeptides, to increase the total mass of the CNP peptides to the rangesdescribed generally herein, e.g., a range from about 2.6 or 2.8 kDa toabout 6 or 7 kDa, can reduce the susceptibility of the modified peptidesto in vivo cleavage by exopeptidases and/or endopeptidases (e.g., NEP).In addition to PEGylation, glycosylation and other chemicalderivatization procedures, e.g., modification by phosphorylation,amidation, carboxylation, acetylation, methylation, and creation ofacid-addition salts, amides, esters and N-acyl derivatives, may alsomask potentially immunogenic regions and/or proteolytically sensitiveregions (Science, 303: 480-482 (2004)).

Examples of chemical modifications include, without limitation, thepolymer addition method of Bednarsaki and the cross-linking method ofAltus Corporation for improving stability and protease resistance andreducing immunogenicity. Bednarsaki showed that polymer addition canimprove protein temperature stability (J. Am. Chem. Soc., 114(1):378-380 (1992)), and Altus Corporation found that glutaraldehydecross-linking can improve enzyme stability.

Chemical modification of polypeptides can be performed in a non-specificfashion (leading to mixtures of derivatized species) or in asite-specific fashion (e.g., based on wild-type macromoleculereactivity-directed derivatization and/or site-selective modificationusing a combination of site-directed mutagenesis and chemicalmodification) or, alternatively, using expressed protein ligationmethods (Curr. Opin. Biotechnol., 13(4): 297-303 (2002)).

Pegylated CNP Variants

In one embodiment, for increased stability (e.g., resistance to NEPdegradation), CNP22 or variants thereof (including those having aminoacid additions, substitutions and/or deletions) are conjugated tohydrophilic, natural or synthetic polymers, to increase the total massof the modified CNP peptides to a range from about 2.6 kDa or 2.8 kDa toabout 4, 5, 6, 7 or higher kDa. In certain embodiments, the addedhydrophilic polymers have a total mass of about 0.6, 0.8, 1, 1.2, 1.4,1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4,4.6, 4.8, or about 5 kDa.

In an embodiment, the hydrophilic polymers are water-soluble so that theCNP peptides conjugated thereto do not precipitate out in an aqueous(e.g., physiological) environment. Further, the hydrophilic polymers arebiocompatible, i.e., do not cause injury, toxicity or an immunologicalreaction in vivo.

The hydrophilic polymers can be branched or unbranched. In oneembodiment, the hydrophilic polymers are not branched.

Various sites of conjugation of CNP22 or variants thereof to ahydrophilic polymer are possible, including but not limited to: (1) onlyat the N-terminus; (2) only at the C-terminus; (3) only at an internalsite (e.g., Lys4); (4) at both the N-terminus and the C-terminus; (5) atthe N-terminus and an internal site; and (6) at the C-terminus and aninternal site. In one embodiment, CNP22 or variants thereof areconjugated to a hydrophilic polymer only at the N-terminus. In anotherembodiment, conjugation is only at an internal site (e.g., Lys4). In yetanother embodiment, conjugation is at the N-terminus and an internalsite (e.g., Lys4). In still another embodiment, for better functionalitythe CNP peptides are not conjugated to a hydrophilic polymer at a site(e.g., Lys10) within the cyclic domain (corresponding to Cys6 to Cys22of CNP22). If conjugation to a hydrophilic polymer is based on bondformation with a reactive primary amino group on the CNP peptide,conjugation at an internal site (e.g., Lys4 and/or Lys10) can beprevented by substitution of Lys4 and/or Lys10 with a natural orunnatural amino acid or peptidomimetic that does not contain a reactiveprimary amino group on a side chain, such as, e.g., Gly, Ser, Arg, Asn,Gln, Asp, Glu or citrulline (Cit). In a particular embodiment, Lys4and/or Lys10 are replaced with Arg. In another embodiment, Lys10 is notreplaced with Arg.

Non-limiting examples of hydrophilic polymers include polymers formedfrom carboxylic acid-bearing monomers (e.g., methacrylic acid (MA) andacrylic acid (AA)), polyvinyl alcohols, polymers formed fromhydroxyl-bearing monomers (e.g., hydroxyethyl methacrylate (HEMA),hydroxypropyl methacrylate (HPMA), hydroxypropyl methacrylamide, and3-trimethylsilylpropyl methacrylate (TMSPMA)), polyalkylene oxides,polyoxyethylated polyols (e.g., glycerol), poly(ethylene glycol) (PEG),poly(propylene glycol), mono-C₁-C₁₀ alkoxy-PEGs (e.g., monomethoxy-PEG),tresyl monomethoxy-PEG, aryloxy-PEGs, PEG acrylate (PEGA), PEGmethacrylate, PEG propionaldehyde, bis-succinimidyl carbonate PEG,copolymers of 2-methacryloyloxyethyl-phosphorylcholine (MPC) and N-vinylpyrrolidone (VP), hydroxy functional poly(N-vinyl pyrrolidone) (PVP),SIS-PEG (SIS is polystyrene-polyisobutylene-polystyrene blockcopolymer), polystyrene-PEG, polyisobutylene-PEG, PCL-PEG (PCL ispolycaprolactone), PLA-PEG (PLA is polylactic acid), PMMA-PEG (PMMA ispoly(methyl methacrylate)), PDMS-PEG (PDMS is polydimethyloxanone),PVDF-PEG (PVDF is polyvinylidene fluoride), PLURONIC™ surfactants(polypropylene oxide-co-polyethylene glycol), poly(tetramethyleneglycol), poly(L-lysine-g-ethylene glycol) (PLL-g-PEG),poly(L-lysine-g-hyaluronic acid) (PLL-g-HA), poly(L-lysine-g-phosphorylcholine) (PLL-g-PC), poly(L-lysine-g-vinyl pyrrolidone) (PLL-g-PVP),poly(ethylimine-g-ethylene glycol) (PEI-g-PEG),poly(ethylimine-g-hyaluronic acid) (PEI-g-HA),poly(ethylimine-g-phosphoryl choline) (PEI-g-PC),poly(ethylimine-g-vinyl pyrrolidone) (PEI-g-PVP), PLL-co-HA, PLL-co-PC,PLL-co-PVP, PEI-co-PEG, PEI-co-HA, PEI-co-PC, PEI-co-PVP, cellulose andderivatives thereof (e.g., hydroxyethyl cellulose), dextran, dextrins,hyaluronic acid and derivatives thereof (e.g., sodium hyaluronate),elastin, chitosan, acrylic sulfate, acrylic sulfonate, acrylicsulfamate, methacrylic sulfate, methacrylic sulfonate, methacrylicsulfamate, polymers and copolymers thereof, and polymers and copolymersof combinations thereof.

In a particular embodiment, the hydrophilic polymer is poly(ethyleneglycol) (PEG), also called poly(ethylene oxide) (PEO). As used herein,the term “PEG” or “PEO” encompasses all the forms of PEG, branched andunbranched, which can be used to derivatize polypeptides, includingwithout limitation mono-(C₁-C₁₀) alkoxy-PEGs and aryloxy-PEGs.

In one embodiment, the PEG-CNP conjugates comprise a PEG (or PEO)polymer of the formula (CH₂CH₂O)_(n), wherein n is an integer from about6 to about 100, and the PEG polymer is from about 0.3 kDa to about 5kDa. In another embodiment, n is an integer from about 12 to about 50,and the PEG polymer is from about 0.6 kDa to about 2.5 kDa. In yetanother embodiment, n is from about 12 to about 24, and the PEG polymeris from about 0.6 kDa to about 1.2 kDa. In a further embodiment, theterminal hydroxyl group of the PEG polymer is capped with a non-reactivegroup. In a particular embodiment, the end-capping group is an alkylgroup, e.g., a lower alkyl group such as methyl, so that the PEG polymerterminates in an alkoxy group. In an embodiment, the PEG polymer is notbranched. In another embodiment, CNP22 or variants thereof areconjugated to a PEG polymer only at the N-terminus.

PEGs and PEOs potentially include molecules with a distribution ofmolecular weights, i.e., they are potentially polydispersed, dependingon the manner in which they are prepared. The size/mass distribution ofa polymeric preparation can be characterized statistically by its weightaverage molecular weight (M_(w)) and its number average molecular weight(M_(n)), the ratio of which is called the polydispersity index(M_(w)/M_(n)). M_(w) and M_(n) can be measured by mass spectroscopy.PEG-CNP variants conjugated to a PEG moiety larger than 1.5 kDa mayexhibit a range of molecular weights due to the polydispersed nature ofthe parent PEG molecule. For example, in the case of mPEG2K (SunbrightME-020HS, NOF Co.), the molecular masses of the PEG molecules aredistributed over a range from about 1.5 kDa to about 3 kDa, with apolydispersity index of 1.036. By contrast, the PEGs conjugated to CNP22or variants thereof using MS (PEG)_(n) reagents (n=4, 8, 12 or 24,denoted as, e.g., “PEO12” or “PEO24”) from Pierce Biotechnology(Rockford, Ill.) are monodispersed, having discrete chain length anddefined molecular weight.

Methods for generating polypeptides comprising a PEG moiety are known inthe art (see, e.g., U.S. Pat. No. 5,824,784). Methods for preparingPEGylated CNP peptides generally comprise the steps of (a) reactingCNP22 or a variant thereof with a PEGylation reagent under conditionssuitable for attaching PEG to the CNP peptide (e.g., at the N-terminus),and (b) obtaining the reaction product(s). Because PEGylating a CNPpeptide might significantly alter its binding to NPR-B, depending on thesize of the PEG moiety and the location of PEGylation, different kindsof PEG and PEGylation reaction conditions can be explored. The chemistrythat can be used for PEGylation of a CNP peptide includes acylation ofreactive primary amine(s) of the peptide using the NHS-ester ofmethoxy-PEG(O—[(N-Succinimidyloxycarbonyl)-methyl]-O′-methylpolyethylene glycol).Acylation with methoxy-PEG-NHS or methoxy-PEG-SPA results in an amidelinkage that eliminates any charge of the original primary amine.PEG-CNP peptides designated with the symbol “PEO12” or “PEO24”, as wellas those designated with the symbol “PEG1K”, “PEG2K”, “PEG5K” or“PEG20K”, are PEGylated via reaction of a primary amino group on thepeptide with an NHS ester-activated, methoxy-end capped PEG reagent.PEG-CNP variants can also be prepared by other methods, e.g., viareductive amination involving a primary amino group on the peptide and aPEG aldehyde, such as, e.g., PEG-propionaldehyde, or mono-C₁-C₁₀ alkoxyor aryloxy derivatives thereof (see U.S. Pat. No. 5,252,714).

Unlike ribosome protein synthesis, synthetic peptide synthesis proceedsfrom the C-terminus to the N-terminus. Accordingly, Boc-PEG (containingtert-butyloxycarbonyl (Boc)) is one method to attach PEG to theC-terminus of a peptide (R. B. Merrifield, J. Am. Chem. Soc., 85(14):2149-2154 (1963)). Alternatively, Fmoc (fluorenylmethoxycarbonyl)chemistry can be employed (E. Atherton and R. C. Sheppard, Solid PhasePeptide Synthesis: a Practical Approach, IRL Press (Oxford, England(1989)).

The present methods for preparing PEG-CNP variants provide asubstantially homogenous mixture of polymer-protein conjugates. Afterpurification, discrete PEG-CNP preparations are sufficiently pure for invitro and in vivo testing of biological properties. As demonstratedherein, certain PEG-CNP variants exhibit reduced susceptibility to NEPcleavage and substantially similar or better functionality (e.g.,stimulation of cGMP production).

As described herein, PEGylation reactions of CNP22 or variants thereof,using appropriate PEGylation reagent/CNP peptide ratios and reactionconditions, provide PEG-CNP derivatives. The nature and extent ofPEGylation can be determined using, e.g., PAGE and HPLC analysis. Incertain embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95% or 99%of CNP22 or variants thereof are mono-PEGylated at the N-terminus. Tooptimize the beneficial effects of PEGylation on the biologicalproperties of a CNP peptide, the polymer length, conformation (e.g.,branched or linear), and/or functionalization (e.g., adding a negativelycharged group) of a PEG moiety can be varied. PEGylated CNP variants aretested for NEP resistance, pharmacokinetics and bioactivity (e.g., theability to bind to NPR-B and stimulate the generation of cGMP).PEGylated CNP variants that show improved NEP resistance and at leastabout 50% of the cGMP-stimulating activity of CNP22 can be furthertested, e.g., in vitro in a rat chondrosarcoma cell-based achondroplasiamodel and in vivo in a murine achondroplasia animal model.

E. Methods of Using CNP Variants, Pharmaceutical Compositions of CNPVariants, and Routes of Administration

Methods of Using CNP Variants

Bone-Related Disorders

Fibroblast growth factors (FGFs) play important roles in bone formation,and mutations in FGF receptor genes (FGFR 1, 2 and 3) give rise to avariety of inherited skeletal malformations (Curr. Biol., 5: 500-507(1995)). In particular, activating mutations in FGFR-3 are responsiblefor disorders of the long bones, including achondroplasia, the mostcommon form of human genetic dwarfism (Nature, 371: 252-254 (1994);Cell, 78: 335-342 (1994)), the milder disorder hypochondroplasia (Ann.N.Y. Acad. Sci., 785: 182-187 (1996)), and the more severe and neonatallethal thanatophoric dysplasia (TD) types I and II (Hum. Mol. Genet., 5:509-512 (1996); Nat. Genet., 9: 321-328 (1995)). Mouse modelsoverexpressing FGF-2, and consequentially activating FGFR-3, showshortened long bones and macrocephaly (Mol. Biol. Cell, 6: 1861-73(1995)). Consistent with this model, mice deficient in FGFR-3 showremarkable skeletal overgrowth with wider growth plates (Nature Genet.,12: 390-397 (1996)).

Complementary experiments with CNP, NPR-B and NPR-C suggest a linkbetween the peptide ligand, the corresponding receptors, and bonegrowth. Activation of NPR-B by elevated plasma concentrations of CNP intransgenic mice causes skeletal overgrowth (Nat. Med., 10: 80-86 (2004))histologically similar to that of the growth plate cartilage of FGFR-3knockout mice (Nat. Genet., 4: 390-397 (1996)). In NPR-C knockout mice,NPR-C-mediated clearance of CNP should be eliminated; consistent withthis prediction, the knockout animals show elongated long bones andelongated vertebrae with kyphosis (Proc. Natl. Acad. Sci. USA 96:7403-08 (1999)). Conversely, CNP knockout mice are dwarfed with shorterlong bones and vertebrae, a phenotype histologically similar to that ofachondroplasia, and have increased mortality as a result of malocclusionand pulmonary restriction from the small rib cage (Proc. Natl. Acad.Sci. USA, 98: 4016-4021 (2001)). Consistent with the proposed role ofCNP as an activator of NPR-B, the NPR-B knockout mouse has the samedwarfed skeletal phenotype and increased mortality as the CNP knockoutmouse (Proc. Natl. Acad. Sci. USA, 101: 17300-05 (2004)). Furthermore,in a mouse model of achondroplasia with activated FGFR-3 in thecartilage, targeted overexpression of CNP in chondrocytes counteractsdwarfism (Yasoda et al., Nat. Med., 10: 80-86 (2004)). Additionally, CNPhas been show to play a role in regulating endochondral bone growth andchondrocyte activity, including but not limited to chondrocyteproliferation and differentiation, inhibition of the mitogen activatedprotein (MAP) kinase/MEK (Raf-1) kinase signaling pathway, and promotionof endochondral ossification (Yasoda et al., Nat. Med., 10: 80-86(2004)). These results suggest that activation of the CNP/NPR-B systemis a potential therapeutic strategy for treatment of humanachondroplasia.

By stimulating matrix production, proliferation and differentiation ofchondrocytes and increasing long bone growth, the CNP variants of thedisclosure are useful for treating mammals, including humans, sufferingfrom a bone-related disorder, such as a skeletal dysplasia. Non-limitingexamples of CNP-responsive bone-related disorders and skeletaldysplasias include achondroplasia, hypochondroplasia, short stature,dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesisimperfecta, achondrogenesis, chondrodysplasia punctata, homozygousachondroplasia, chondrodysplasia punctata, camptomelic dysplasia,congenital lethal hypophosphatasia, perinatal lethal type ofosteogenesis imperfecta, short-rib polydactyly syndromes,hypochondroplasia, rhizomelic type of chondrodysplasia punctata,Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasiacongenita, atelosteogenesis, diastrophic dysplasia, congenital shortfemur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelicdysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis,peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Robertssyndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniestsyndrome, metatrophic dysplasia, and spondyloepimetaphyseal dysplasia.Further, the CNP variants are useful as an adjunct or alternative togrowth hormone for treating idiopathic short stature and other skeletaldysplasias.

In addition, the CNP variants are useful for treating other bone-relatedconditions and disorders, such as rickets, hypophosphatemic rickets[including X-linked hypophosphatemic rickets (also called vitaminD-resistant rickets) and autosomal dominant hypophosphatemic rickets],and osteomalacia [including tumor-induced osteomalacia (also calledoncogenic osteomalacia or oncogenic hypophosphatemic osteomalacia)].

The CNP variants of the disclosure can also be used to treatosteoarthritis. Osteoarthritis is a degenerative disease of thearticular cartilage and occurs frequently in the elderly. Osteoarthritisinvolves destruction of the cartilage and proliferative change in thebone and cartilage resulting from degeneration of articular components,with the change resulting in a secondary arthritis (e.g., synovitis).The extracellular matrix proteins, which are the functional entity ofthe cartilage, are reduced, and the number of chondrocytes decreases inosteoarthritis (Arth. Rheum. 46(8): 1986-1996 (2002)). By promoting thematrix production, growth and differentiation of chondrocytes, the CNPvariants are useful for countering the undesired effects of FGF-2 andincreasing matrix synthesis in subjects suffering from arthritis,including osteoarthritis, thereby treating arthritis, includingosteoarthritis.

Vascular Smooth Muscle Disorders

CNP and other vasoactive peptides (including ANP, BNP and urodilatin)have vasodilator and diuretic properties and play an important role incardiovascular homeostasis (J. Cardiovasc. Pharmacol., 117: 1600-06(1998); Kidney Int., 49: 1732-37 (1996); Am. J. Physiol., 275:H1826-1833 (1998)). CNP is widely distributed in the cardiovascularsystem, especially in high concentration in vascular endothelial cells(J. Cardiovasc. Pharmacol., 117: 1600-06 (1998)). CNP is a potentrelaxant of vascular smooth muscle, particularly in the coronarycirculation (Biochem. Biophys. Res. Commun., 205: 765-771 (1994)), andis an inhibitor of smooth muscle cell proliferation (Biochem. Biophys.Res. Commun., 177: 927-931 (1991)). Although the vasodilator effect ofCNP is less potent than that of ANP (about 1:100) (Hypertens. Res., 21:7-13 (1998); Am. J. Physiol., 275: L645-L652 (1998)), CNP mRNA isincreased in response to shear stress (FEBS Lett., 373: 108-110 (1995))and plasma levels of CNP are elevated in inflammatory cardiovascularpathologies (Biochem. Biophys. Res. Commun., 198: 1177-1182 (1994)). CNPhas been shown to suppress inflammation through inhibition of macrophageinfiltration in injured carotid arteries of rabbits (Circ. Res., 91:1063-1069 (2002)) and to directly inhibit cardiac fibroblastproliferation through an NPR-B/cGMP-dependent pathway (Endocrinology,144: 2279-2284 (2003)).

The cardiovascular actions of CNP are mediated via activation of the NPRsubtypes, NPR-B and NPR-C (Endocrinology, 130: 229-239 (1992)), thelatter accounting for 95% of NPRs expressed in vivo (Science, 293:1657-1662 (2001)). The CNP/NPR-B pathway leads to elevation of cGMP, awell-established secondary messenger in the cardiovascular system.NPR-C's 37-amino acid portion from the C-terminus has a consensussequence that interacts with the heterotrimeric G protein G_(i) (J.Biol. Chem., 274: 17587-17592 (1999)), which has been shown to regulateadenylate cyclase and phospholipase C activity (J. Biol. Chem., 276:22064-70 (2001); Am. J. Physiol., 278: G974-980 (2000); J. Biol. Chem.,271: 19324-19329 (1996)). CNP mediates smooth muscle hyperpolarizationand relaxation via activation of NPR-C and the opening of a Gprotein-regulated, inwardly rectifying K⁺ channels (Proc. Natl. Acad.Sci. USA, 100: 1426-1431 (2003)). Likewise, CNP has importantanti-proliferative effects in cardiac fibroblasts and, throughinteraction with NPR-C, regulates local blood flow and systemic bloodpressure by hyperpolarizing smooth muscle cells (R. Rose and W. Giles,J. Physiol. 586: 353-366 (2008)).

By binding to NPR-B on vascular smooth muscle cells, CNP22 stimulatesthe production of cGMP, which acts as an intracellular secondarymessenger to cause ultimately the relaxation of blood vessels. Based onthe hypotensive actions of CNP, the CNP variants of the disclosure areuseful for treating hypertension, congestive heart failure, cardiacedema, nephredema, hepatic edema, acute and chronic renal insufficiency,and so on. In addition, activation of cGMP signaling suppresses thegrowth of vascular smooth muscle cells. Accordingly, the CNP variants ofthe disclosure can be used to treat conditions or diseases caused by theabnormal growth of vascular smooth muscle cells, including but notlimited to restenosis and arteriosclerosis.

The studies described above suggest that CNP may be a potentialtherapeutic candidate for vascular smooth muscle relaxation andremodeling. Pharmacological effects of CNP concerning certain disordershave been attributed, in part, to vasoprotective effects rather than tovasodilator activity (Am. J. Respir. Crit. Care Med., 170: 1204-1211(2004)). Therefore, the CNP variants of the present disclosure areuseful for treating conditions, e.g., vascular smooth muscle disorders,in which CNP may have a vasoprotective effect, including withoutlimitation inducing smooth muscle relaxation and inhibiting infiltrationof macrophages into cardiac tissue. In one embodiment, the CNP variantsare used to treat heart failure, including but not limited to acutedecompensated heart failure and acute congestive heart failure. Inanother embodiment, the CNP variants are used to treat asthma,cardiomyopathy, and restenosis of coronary arteries (by increasingsmooth muscle cell relaxation and decreasing proliferation of smoothmuscle cells).

Pharmaceutical Compositions of CNP Variants

In additional embodiments, the disclosure provides pharmaceuticalcompositions comprising a CNP variant, and one or more pharmaceuticallyacceptable excipients, carriers and/or diluents. In certain embodiments,the compositions further comprise one or more other biologically activeagents (e.g., inhibitors of proteases, receptor tyrosine kinases, and/orthe clearance receptor NPR-C).

In some embodiments, the compositions comprise the desired CNP variantin at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%purity. In certain embodiments, the compositions contain less than about10%, 5%, 4%, 3%, 2%, 1% or 0.5% of macromolecular contaminants, such asother mammalian (e.g., human) proteins and other CNP variants.

Non-limiting examples of excipients, carriers and diluents includevehicles, liquids, buffers, isotonicity agents, additives, stabilizers,preservatives, solubilizers, surfactants, emulsifiers, wetting agents,adjuvants, and so on. The compositions can contain liquids (e.g., water,ethanol); diluents of various buffer content (e.g., Tris-HCl, phosphate,acetate buffers, citrate buffers), pH and ionic strength; detergents andsolubilizing agents (e.g., Polysorbate 20, Polysorbate 80);anti-oxidants (e.g., methionine, ascorbic acid, sodium metabisulfite);preservatives (e.g., Thimerosol, benzyl alcohol, m-cresol); and bulkingsubstances (e.g., lactose, mannitol, sucrose). The use of excipients,diluents and carriers in the formulation of pharmaceutical compositionsis known in the art; see, e.g., Remington's Pharmaceutical Sciences,18^(th) Edition, pages 1435-1712, Mack Publishing Co. (Easton, Pa.(1990)), which is incorporated herein by reference in its entirety.

For example, carriers include without limitation diluents, vehicles andadjuvants, as well as implant carriers, and inert, non-toxic solid orliquid fillers and encapsulating materials that do not react with theactive ingredient(s). Non-limiting examples of carriers includephosphate buffered saline, physiological saline, water, and emulsions(e.g., oil/water emulsions). A carrier can be a solvent or dispersingmedium containing, e.g., ethanol, a polyol (e.g., glycerol, propyleneglycol, liquid polyethylene glycol, and the like), a vegetable oil, andmixtures thereof.

In some embodiments, the compositions are liquid formulations. Incertain embodiments, the formulations comprise a CNP variant in aconcentration range from about 0.1 mg/ml to about 20 mg/ml, or fromabout 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/ml to about 20mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10 mg/ml.

In further embodiments, the compositions comprise a buffer solution orbuffering agent to maintain the pH of a CNP-containing solution orsuspension within a desired range. Non-limiting examples of buffersolutions include phosphate buffered saline, Tris buffered saline, andHank's buffered saline. Buffering agents include without limitationsodium acetate, sodium phosphate, and sodium citrate. Mixtures ofbuffering agents can also be used. In certain embodiments, the bufferingagent is acetic acid/acetate or citric acid/citrate. The amount ofbuffering agent suitable in a composition depends in part on theparticular buffer used and the desired pH of the solution or suspension.For example, acetate is a more efficient pH buffer at pH 5 than pH 6, soless acetate may be used in a solution at pH 5 than at pH 6. In someembodiments, the buffering agent has a concentration of about 10 mM±5mM. In certain embodiments, the pH of a composition is from about pH 3to about pH 7.5, or from about pH 3.5 to about pH 7, or from about pH3.5 to about pH 6.5, or from about pH 4 to about pH 6, or from about pH4 to about pH 5, or is at about pH 5.0±1.0.

In other embodiments, the compositions contain an isotonicity-adjustingagent to render the solution or suspension isotonic and more compatiblefor injection. Non-limiting examples of isotonicity agents include NaCl,dextrose, glucose, glycerin, sorbitol, xylitol, and ethanol. In certainembodiments, the isotonicity agent is NaCl. In certain embodiments, NaClis in a concentration of about 160±20 mM, or about 140 mM±20 mM, orabout 120±20 mM, or about 100 mM±20 mM, or about 80 mM±20 mM, or about60 mM±20 mM.

In yet other embodiments, the compositions comprise a preservative.Preservatives include, but are not limited to, m-cresol and benzylalcohol. In certain embodiments, the preservative is in a concentrationof about 0.4%±0.2%, or about 1%±0.5%, or about 1.5%±0.5%, or about2.0%±0.5%.

In still other embodiments, the compositions contain an anti-adsorbent(e.g., to mitigate adsorption of a CNP variant to glass or plastic).Anti-adsorbents include without limitation benzyl alcohol, Polysorbate20, and Polysorbate 80. In certain embodiments, the anti-adsorbent is ina concentration from about 0.001% to about 0.5%, or from about 0.01% toabout 0.5%, or from about 0.1% to about 1%, or from about 0.5% to about1%, or from about 0.5% to about 1.5%, or from about 0.5% to about 2%, orfrom about 1% to about 2%.

In additional embodiments, the compositions comprise a stabilizer.Non-limiting examples of stabilizers include glycerin, glycerol,thioglycerol, methionine, and ascorbic acid and salts thereof. In someembodiments, when the stabilizer is thioglycerol or ascorbic acid or asalt thereof, the stabilizer is in a concentration from about 0.1% toabout 1%. In other embodiments, when the stabilizer is methionine, thestabilizer is in a concentration from about 0.01% to about 0.5%, or fromabout 0.01% to about 0.2%. In still other embodiments, when thestabilizer is glycerin, the stabilizer is in a concentration from about5% to about 100% (neat).

In further embodiments, the compositions contain an antioxidant.Exemplary anti-oxidants include without limitation methionine andascorbic acid. In certain embodiments, the molar ratio of antioxidant toCNP variant is from about 0.1:1 to about 15:1, or from about 1:1 toabout 15:1, or from about 0.5:1 to about 10:1, or from about 1:1 toabout 10:1 or from about 3:1 to about 10:1.

Pharmaceutically acceptable salts can be used in the compositions,including without limitation mineral acid salts (e.g., hydrochloride,hydrobromide, phosphate, sulfate), salts of organic acids (e.g.,acetate, propionate, malonate, benzoate, mesylate, tosylate), and saltsof amines (e.g., isopropylamine, trimethylamine, dicyclohexylamine,diethanolamine). A thorough discussion of pharmaceutically acceptablesalts is found in Remington's Pharmaceutical Sciences, 18^(th) Edition,Mack Publishing Company, (Easton, Pa. (1990)).

The pharmaceutical compositions can be administered in various forms,such as tablets, capsules, granules, powders, solutions, suspensions,emulsions, ointments, and transdermal patches. The dosage forms of thecompositions can be tailored to the desired mode of administration ofthe compositions. For oral administration, the compositions can take theform of, e.g., a tablet or capsule (including softgel capsule), or canbe, e.g., an aqueous or nonaqueous solution, suspension or syrup.Tablets and capsules for oral administration can include one or morecommonly used excipieints, diluents and carriers, such as mannitol,lactose, glucose, sucrose, starch, corn starch, sodium saccharin, talc,cellulose, magnesium carbonate, and lubricating agents (e.g., magnesiumstearate, sodium stearyl fumarate). If desired, flavoring, coloringand/or sweetening agents can be added to the solid and liquidformulations. Other optional ingredients for oral formulations includewithout limitation preservatives, suspending agents, and thickeningagents. Oral formulations can also have an enteric coating to protectthe CNP variant from the acidic environment of the stomach. Methods ofpreparing solid and liquid dosage forms are known, or will be apparent,to those skilled in this art (see, e.g., Remington's PharmaceuticalSciences, referenced above).

Formulations for parenteral administration can be prepared, e.g., asliquid solutions or suspensions, as solid forms suitable forsolubilization or suspension in a liquid medium prior to injection, oras emulsions. For example, sterile injectable solutions and suspensionscan be formulated according to techniques known in the art usingsuitable diluents, carriers, solvents (e.g., buffered aqueous solution,Ringer's solution, isotonic sodium chloride solution), dispersingagents, wetting agents, emulsifying agents, suspending agents, and thelike. In addition, sterile fixed oils, fatty esters, polyols and/orother inactive ingredients can be used. As further examples,formulations for parenteral administration include aqueous sterileinjectable solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions, which can contain suspending agents and thickening agents.

Compositions comprising a CNP variant can also be lyophilizedformulations. In certain embodiments, the lyophilized formulationscomprise a buffer and bulking agent, and optionally an antioxidant.Exemplary buffers include without limitation acetate buffers and citratebuffers. Exemplary bulking agents include without limitation mannitol,sucrose, dexran, lactose, trehalose, and povidone (PVP K24). In certainembodiments, mannitol is in an amount from about 3% to about 10%, orfrom about 4% to about 8%, or from about 4% to about 6%. In certainembodiments, sucrose is in an amount from about 6% to about 20%, or fromabout 6% to about 15%, or from about 8% to about 12%. Exemplaryanti-oxidants include, but are not limited to, methionine and ascorbicacid.

The disclosure also provides kits containing, e.g., bottles, vials,ampoules, tubes, cartridges and/or syringes that comprise a liquid(e.g., sterile injectable) formulation or a solid (e.g., lyophilized)formulation. The kits can also contain pharmaceutically acceptablevehicles or carriers (e.g., solvents, solutions and/or buffers) forreconstituting a solid (e.g., lyophilized) formulation into a solutionor suspension for administration (e.g., by injection), including withoutlimitation reconstituting a lyophilized formulation in a syringe forinjection or for diluting concentrate to a lower concentration.Furthermore, extemporaneous injection solutions and suspensions can beprepared from, e.g., sterile powder, granules, or tablets comprising aCNP-containing composition. The kits can also include dispensingdevices, such as aerosol or injection dispensing devices, pen injectors,autoinjectors, needleless injectors, syringes, and/or needles.

As a non-limiting example, a kit can include syringes having a singlechamber or dual chambers. For single-chamber syringes, the singlechamber can contain a liquid CNP formulation ready for injection, or asolid (e.g., lyophilized) CNP formulation or a liquid formulation of aCNP variant in a relatively small amount of a suitable solvent system(e.g., glycerin) that can be reconstituted into a solution or suspensionfor injection. For dual-chamber syringes, one chamber can contain apharmaceutically acceptable vehicle or carrier (e.g., solvent system,solution or buffer), and the other chamber can contain a solid (e.g.,lyophilized) CNP formulation or a liquid formulation of a CNP variant ina relatively small amount of a suitable solvent system (e.g., glycerin)which can be reconstituted into a solution or suspension, using thevehicle or carrier from the first chamber, for injection.

As a further example, a kit can include one or more pen injector orautoinjector devices, and dual-chamber cartridges. One chamber of acartridge can contain a pharmaceutically acceptable vehicle or carrier(e.g., solvent system, solution or buffer), and the other chamber cancontain a solid (e.g., lyophilized) CNP formulation or a liquidformulation of a CNP variant in a relatively small amount of a suitablesolvent system (e.g., glycerin) which can be reconstituted into asolution or suspension, using the vehicle or carrier from the firstchamber, for injection. A cartridge can comprise an amount of the CNPvariant that is sufficient for dosing over a desired time period (e.g.,1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, etc.). The peninjector or autoinjector can be adjusted to administer a desired amountof the CNP formulation from a cartridge.

In addition, pharmaceutical compositions comprising a CNP variant can beformulated as a slow release, controlled release or sustained releasesystem for maintaining a relatively constant level of dosage over adesired time period, such as 1 week, 2 weeks, 3 weeks, 1 month, 2months, or 3 months. Slow release, controlled release and sustainedrelease formulations can be prepared using, e.g., biodegradablepolymeric systems {which can comprise, e.g., hydrophilic polymers [e.g.,polylactide, polyglycolide, poly(lactide-glycolide)]}, and can take theform of, e.g., microparticles, microspheres or liposomes, as is known inthe art.

Dosages and Frequency of Dosing

As used herein, the term “therapeutically effective amount” of an activeagent (e.g., a CNP variant) refers to an amount that providestherapeutic benefit to a patient. The amount may vary from oneindividual to another and may depend upon a number of factors, includingthe overall physical condition of the patient. A therapeuticallyeffective amount of a CNP variant can be readily ascertained by oneskilled in the art, using publicly available materials and procedures.For example, the amount of a CNP variant used for therapy should give anacceptable rate of growth based on growth charts for children ages 0-17years with achondroplasia (214 females and 189 males), which list heightfor age, head circumference, and segmental growth (Horton W A et al.,Standard growth curves for achondroplasia, J. Pediatr., 93: 435-8(1978)). CDC charts can be used to assess weight for age and weight forheight or BMI for age. Secondary outcomes with courses that are morechronic in nature can also be measured.

Having a longer serum half-life than wild-type CNP22, the CNP variantscan potentially be administered less frequently than CNP22. The dosingfrequency for a particular individual may vary depending upon variousfactors, including the disorder being treated and the condition andresponse of the individual to the therapy. In certain embodiments, apharmaceutical composition containing a CNP variant is administered to asubject about one time per day, one time per two days, one time perthree days, or one time per week. In one embodiment, for treatment ofbone-related disorders (e.g., skeletal dysplasias, includingachondroplasia), a daily or weekly dose of a CNP variant is administeredto patients until and/or through adulthood.

The CNP variants described herein can be administered to patients attherapeutically effective doses to treat, ameliorate or preventbone-related disorders (e.g., skeletal dysplasias, includingachondroplasia) and conditions (e.g., vascular smooth muscle disorders)in which CNP can provide a vasoprotective effect. The safety andtherapeutic efficacy of the CNP variants can be determined by standardpharmacological procedures in cell cultures or experimental animals,such as, for example, by determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀ (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Active agents exhibiting a large therapeutic index are normallypreferred.

Data obtained from cell culture assays and animal studies can be used toformulate a range of dosage for use in humans. The dosage normally lieswithin a range of circulating concentrations that include the ED₅₀, withlittle or minimal toxicity. The dosage can vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The therapeutically effective dose can be determined from cellculture assays and animal studies.

In certain embodiments, the CNP variants described herein areadministered at a dose in the range from about 5 or 10 nmol/kg to about300 nmol/kg, or from about 20 nmol/kg to about 200 nmol/kg. In someembodiments, the CNP variants are administered at a dose of about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 750, 1000,1250, 1500, 1750 or 2000 nmol/kg or other dose deemed appropriate by thetreating physician. In other embodiments, the CNP variants areadministered at a dose of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000ug/kg, or about 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5 or 10 mg/kg, or other dose deemed appropriate by thetreating physician. The doses of CNP variants described herein can beadministered according to the dosing frequency/frequency ofadministration described herein, including without limitation daily, 2or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc.

The frequency of dosing/administration of a CNP variant for a particularsubject may vary depending upon various factors, including the disorderbeing treated and the condition and response of the subject to thetherapy. The CNP variant can be administered in a single dose or inmultiple doses per dosing. In certain embodiments, the CNP variant isadministered, in a single dose or in multiple doses, daily, every otherday, every 3 days, 2 times per week, 3 times per week, weekly,bi-weekly, every 3 weeks, monthly, every 6 weeks, every 2 months, every3 months, or as deemed appropriate by the treating physician.

In some embodiments, a CNP variant is administered so as to allow forperiods of growth (e.g., chondrogenesis), followed by a recovery period(e.g., osteogenesis). For example, the CNP variant may be administeredintravenously, subcutaneously or by another mode daily or multiple timesper week for a period of time, followed by a period of no treatment,then the cycle is repeated. In some embodiments, the initial period oftreatment (e.g., administration of the CNP variant daily or multipletimes per week) is for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12weeks. In a related embodiment, the period of no treatment lasts for 3days, 1 week, 2 weeks, 3 weeks or 4 weeks. In certain embodiments, thedosing regimen of the CNP variant is daily for 3 days followed by 3 daysoff; or daily or multiple times per week for 1 week followed by 3 daysor 1 week off; or daily or multiple times per week for 2 weeks followedby 1 or 2 weeks off; or daily or multiple times per week for 3 weeksfollowed by 1, 2 or 3 weeks off; or daily or multiple times per week for4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks followed by 1, 2, 3 or 4 weeks off.

Modes of Administration

The CNP variants, or pharmaceutical compositions comprising them, can beadministered to subjects in various ways such as, e.g., by injectionsubcutaneously, intravenously, intra-arterially, intraperitoneally,intramuscularly, intradermally, or intrathecally. In an embodiment, theCNP variants are administered by a single subcutaneous, intravenous,intra-arterial, intraperitoneal, intramuscular, intradermal orintrathecal injection once a day.

The CNP variants can also be administered by direct injection at or nearthe site of disease. Further, the CNP variants can be administered byimplantation of a depot at the target site of action (e.g., an abnormalor dysplasic bone). Alternatively, the CNP variants can be administeredsublingually under the tongue (e.g., sublingual tablet) or by inhalationinto the lungs (e.g., inhaler or aerosol spray), by delivery into thenasal cavity (e.g., intranasal spray), by delivery into the eye (e.g.,eye drop), or by transdermal delivery (e.g., by means of a patch on theskin). The CNP variants may also be administered orally in the form ofmicrospheres, microcapsules, liposomes (uncharged or charged (e.g.,cationic)), polymeric microparticles (e.g., polyamides, polylactide,polyglycolide, poly(lactide-glycolide)), microemulsions, and the like.

A further method of administration is by osmotic pump (e.g., an Alzetpump) or mini-pump (e.g., an Alzet mini-osmotic pump), which allows forcontrolled, continuous and/or slow-release delivery of the CNP variantor pharmaceutical composition over a pre-determined period. The osmoticpump or mini-pump can be implanted subcutaneously, or near the targetsite (e.g., the long bones of limbs, the epiphyses, etc.).

As explained above, the CNP variants can be used to treat conditions ordiseases caused by the abnormal growth of vascular smooth muscle cells,including but not limited to restenosis and arteriosclerosis. For localdelivery of a CNP variant to the diseased bodily vessel (e.g., bloodvessel), the CNP variant can be delivered by means of a medical device(e.g., a stent) implanted at the diseased site. In one embodiment, theCNP variant is impregnated in a polymeric matrix or polymeric coatingdisposed over a stent. In another embodiment, the CNP variant iscontained in reservoirs or channels formed in the body of a stent andcovered by a porous polymeric membrane or layer through which the CNPvariant can diffuse. The polymeric matrix, coating, membrane or layercan comprise at least one biodegradable (e.g., hydrophilic) polymer, asis known in the art. In a further embodiment, the CNP variant can becontained in micropores in the body of a stent. The CNP variant can bedelivered from a stent by burst release, pulse release, controlledrelease or sustained release, or a combination thereof. For example, thestent can locally deliver the CNP variant to the diseased site in aburst release followed by a sustained release. Sustained release can beover a period up to about 2 weeks, 1 month, 2 months, 3 months, 6 monthsor 1 year.

It will be apparent to one skilled in the art that the CNP variants orcompositions thereof can also be administered by other modes.Determination of the most effective mode of administration of the CNPvariants or compositions thereof is within the skill of the skilledartisan.

The CNP variants can be administered as pharmaceutical formulationssuitable for, e.g., oral (including buccal and sub-lingual), rectal,nasal, topical, pulmonary, vaginal or parenteral (includingintramuscular, intraarterial, intrathecal, subcutaneous and intravenous)administration, or in a form suitable for administration by inhalationor insufflation. Depending on the intended mode of administration, thepharmaceutical formulations can be in the form of solid, semi-solid orliquid dosage forms, such as tablets, suppositories, pills, capsules,powders, liquids, suspensions, emulsions, creams, ointments, lotions,and the like. The formulations can be provided in unit dosage formsuitable for single administration of a precise dosage. The formulationscomprise an effective amount of a CNP variant, and one or morepharmaceutically acceptable excipients, carriers and/or diluents, andoptionally one or more other biologically active agents.

Combination Therapy

In one embodiment, a CNP variant can be used in combination with one ormore other active agents useful for treating, ameliorating or preventingCNP-responsive conditions or disorders such as, e.g., bone-relateddisorders (e.g., skeletal dysplasias) and vascular smooth muscledisorders. The other active agent(s) can enhance the effects of the CNPvariant and/or exert other pharmacological effects in addition to thoseof the CNP variant. Non-limiting examples of active agents that can beused in combination with the CNP variants described herein are othernatriuretic peptides (e.g., BNP) and inhibitors (e.g., antagonists) ofpeptidases and proteases (e.g., NEP and furin), NPR-C and tyrosinekinases (e.g., FGFR-3). By preventing NEP cleavage of the CNP variant,an NEP inhibitor can prolong the half-life of the variant. Examples ofNEP inhibitors include, without limitation, thiorphan and candoxatril.Co-use of an NPR-C inhibitor can also prolong the half-life of the CNPvariant via inhibition of the variant's clearance by NPR-C. Anon-limiting example of an NPR-C inhibitor is the fragment FGIPMDRIGRNPR(SEQ ID NO: 82), which would be released at the target site (e.g., bonegrowth plate) upon proteolytic cleavage of the FGIPMDRIGRNPR-CNP22chimera (Analog CZ) (SEQ ID NO: 82) or similar chimeras comprisingvariants of CNP22 (e.g., those containing amino acid substitution(s),addition(s), and/or deletion(s) relative to CNP22). Co-use of a tyrosinekinase inhibitor can accentuate the effects of a CNP therapy byinhibiting the tyrosine kinase receptor FGFR-3, a negative regulator ofchondrocyte and bone growth. Non-limiting examples of tyrosine kinaseinhibitors include those disclosed in U.S. Pat. Nos. 6,329,375 and6,344,459.

To achieve the appropriate therapeutic outcome in the combinationtherapies, one would generally administer to the subject the CNPcomposition and other therapeutic(s) in a combined amount effective toproduce the desired therapeutic outcome (e.g., restored bone growth).This process may involve administering the CNP composition and othertherapeutic agent(s) at the same time. Simultaneous administration canbe achieved by administering a single composition or pharmacologicalprotein formulation that includes both the CNP variant and othertherapeutic agent(s). Alternatively, the other therapeutic agent(s) canbe taken separately at about the same time as a pharmacologicalformulation (e.g., tablet, injection or drink) of the CNP variant. TheCNP variant can also be formulated into a foodstuff such as brownies,pancakes, or cake, suitable for ingestion.

In other alternatives, administration of the CNP variant can precede orfollow administration of the other therapeutic agent(s) by intervalsranging from minutes to hours. In embodiments where the othertherapeutic agent(s) and the CNP composition are administeredseparately, one would generally ensure that the CNP variant and theother therapeutic agent(s) are administered within an appropriate timeof one another so that both the CNP variant and the other therapeuticagent(s) can exert, synergistically or additively, a beneficial effecton the patient. For example, one can administer the CNP compositionwithin about 0.5-6 hours (before or after) of the other therapeuticagent(s). In one embodiment, the CNP composition is administered withinabout 1 hour (before or after) of the other therapeutic agent(s).

Identifying and Monitoring Patient Populations

Protocols can be established to identify subjects suitable for CNPtherapy and to determine whether a given patient is responsive to CNPtherapy. For example, for treatment of bone-related disorders,indicators of growth can be measured, such as long bone growthmeasurements in utero and neonatal and measurements of bone growthbiomarkers such as CNP, cGMP, Collagen II, osteocalcin, andProliferating Cell Nuclear Antigen (PCNA).

One CNP signaling marker is cGMP (guanosine 3′,5′ cyclic monophosphate).The level of this intracellular signaling molecule increases after CNPbinds to and activates its cognate receptor NPR-B. Elevated levels ofcGMP can be measured from cell culture extracts (in vitro) after CNPexposure, conditioned media from bone ex-plant studies (ex vivo) afterCNP exposure, and in the plasma (in vivo) within minutes of CNPadministration subcutaneously, intravenously, or via other routes ofadministration known in the art.

Cartilage and bone-specific analytes (or cartilage- and bone-associatedmarkers) can also be measured to assess CNP efficacy. For example,fragments of cleaved collagen type II are a cartilage-specific markerfor cartilage turnover. Type II collagen is the major organicconstituent of cartilage and fragments of type II collagen (cleavedcollagen) are released into circulation, and subsequently secreted intothe urine, following cartilage turnover. Cartilage turnover preceeds newbone formation.

A bone-specific biomarker for bone formation which can be measured isN-terminal propeptides of type I procollagen (PINP). The synthesis oftype I collagen is an important step in bone formation, as type Icollagen is the major organic component in bone matrix. During collagensynthesis, propeptides are released from the procollagen molecule andcan be detected in serum. In addition, fragments of collagen type I canbe measured as a marker for bone resorption.

Other potential biomarkers for cartilage and bone formation and growthinclude aggrecan chondroitin sulfate (cartilage-specific marker forcartilage turnover), propeptides of type TT collagen (cartilage-specificmarker for cartilage formation), alkaline phosphatase (bone-specific)and osteocalcin (bone-specific marker for bone formation). Cartilage-and bone-associated biomarkers can be measured, e.g., in serum fromefficacy/pharmacodynamic in vivo studies and from the conditioned mediaof ex vivo studies, using commercially available kits.

In one embodiment, the level of at least one bone- orcartilage-associated biomarker is assayed or measured in a subject thathas been administered a CNP variant in order to monitor the effects ofthe CNP variant on bone and cartilage formation and growth in vivo. Forexample, an increase in the level of at least one bone- orcartilage-associated biomarker may indicate that administration of a CNPvariant has a positive effect on bone growth and is a useful treatmentfor skeletal dysplasias and other bone- or cartilage-related diseases ordisorders associated with decreased CNP activity. Exemplary bone- orcartilage-associated biomarkers include, but are not limited to, CNP(e.g, endogenous levels of CNP), cGMP, propeptides of collagen type IIand fragments thereof, collagen type II and fragments thereof,osteocalcin, proliferating cell nuclear antigen (PCNA), propeptides oftype I procollagen (PINP) and fragments thereof, collagen type I andfragments thereof, aggrecan chondroitin sulfate, and alkalinephosphatase.

In an embodiment, biomarkers are measured by obtaining a biologicalsample from a subject who will be administered, is being administered orhas been administered a CNP variant. Biomarkers can be measured usingtechniques known in the art, including, but not limited to, WesternBlot, enzyme linked immunosorbant assay (ELISA), and enzymatic activityassay. The biological sample can be blood, serum, urine, or otherbiological fluids.

Additional aspects and details of the disclosure will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting.

F. Examples Example 1 Synthesis of CNP Variants

CNP variants were prepared using the methods described herein.Substitutions with natural or unnatural amino acids or peptidomimeticswere made, as indicated in Tables 1-3 (shown in Example 3), at therespective amino acid residues in the wild-type sequence of CNP22. Incertain variants, additional amino acids were added to the N-terminaland/or C-terminal ends of the whole or a portion of the wild-type CNP22sequence (see Table 3).

Also prepared were CNP variants in which a PEG (or PEO) moiety wasconjugated to the N-terminus of CNP22 or variants thereof (see Table 4,shown in Example 3). PEGylation reagents can be obtained from thecommercial sources shown in Table 5.

TABLE 5 Vendor Product Name Name MW (Da) PEGylation Reagent NOFSunbright ME-200CS mPEG20K 20,000 CH3(CH2CH2O)450- (CH2)5COO—NHS NOFSunbright ME-050CS mPEG5K 5,000 CH3(CH2CH2O)110- (CH2)5COO—NHS Pierce(Methyl-PEG12)3-PEG4-NHS (mPEG12)3-PEG4 2,400 [CH3(CH2CH2O)12]3- Ester(CH2CH2O)4-NHCO(CH2)3- COO—NHS NOF Sunbright ME-020HS mPEG2K 2,000CH3(CH2CH2O)45- (CH2)5COO—NHS NOF Sunbright ME-020CS mPEG2K 2,000CH3(CH2CH2O)45- (CH2)5COO—NHS NOF Sunbright ME-010HS mPEG1K 1,000CH3(CH2CH2O)23- CO(CH2)2COO—NHS Pierce Methyl PEG24-NHS Ester MS(PEG)241,200 CH3(CH2CH2O)24- (CH2)2COO—NHS Pierce EZ-Link NHS-PEG12-BiotinPEO12-Biotin 940 Biotin-(CH2CH2O)12- (CH2)2COO—NHS Pierce MethylPEG12-NHS Ester MS(PEG)12 690 CH3(CH2CH2O)12- (CH2)2COO—NHS PierceEZ-Link NHS-PEG4-Biotin PEO4-biotin 590 Biotin-(CH2CH2O)4- (CH2)2COO—NHSPierce Mono(lactosylamido) LSS 590 mono(succinimidyl)suberate PierceEZ-link NHS-LC-LC-Biotin LC-LC-Biotin 570 Pierce EZ-link NHS-LC-BiotinLC-Biotin 450 (LC = long chain) Pierce EZ-link NHS-Biotin Biotin 340

The PEG (also called PEO) polymers purchased from Pierce Biotechnology(Rockford, Ill.) are monodispersed—i.e., they contain a single discretepolymer of a particular molecular weight. By contrast, the PEG polymerspurchased from NOF (Nippon Oil and Fat) are polydispersed—i.e., theycontain a mixture of polymers having a distribution of molecularweights.

To PEGylate CNP22 or variants thereof, reaction and purificationconditions are optimized for each PEG-CNP conjugate. According to ageneral PEGylation procedure, reaction mixtures contain about 1 mM CNP22or a variant thereof, and about 1 to 5 mM NHS-activated PEG in potassiumphosphate buffer, pH between about 5.0 and 6.5. To mono-PEGylateselectively at the peptide N-terminus and minimize PEGylation at aninternal site (e.g., Lys4 of CNP22), the PEGylation reaction can beconducted under more acidic conditions (e.g., at a pH between about 5.5and 6.5) to protonate selectively and hence deactivate the more basicprimary amino group on the lysine side chain. After about 1 to 3 hoursof incubation at room temperature, the PEGylation reaction is quenchedby addition of aqueous glycine buffer. Reaction products are thenseparated by reverse-phase HPLC, optimized for each PEG-CNP conjugate.Fractionation samples are speedvacced to dryness, andreconstituted/formulated in 1 mM HCl. Identification and purity of eachPEG-CNP product are determined by liquid chromatography-massspectrometry (LC/MS).

Example 2A Recombinant Production of CNP Variants

CNP variants can be produced using recombinant technology. In certainembodiments, the CNP variants are produced as fusion proteins comprisinga cleavable peptide, carrier protein or tag. Exemplary methods forrecombinantly producing CNP fusion proteins are disclosed below.

Materials and Methods

Cloning of CNP Fusion Proteins into Expression Vectors

CNP DNA fragments were amplified using polymerase chain reaction (PCR)and the amplified PCR fragments were digested with Nde I and BamHI andcloned into pET21a vector (Novagen, Gibbstown, N.J.). CNP fusion proteinDNA was synthesized by DNA2.0 and cloned into different expressionvectors (Table 6).

TABLE 6 Expression Chemical Final E. coli Construct Vector Productcleavage product strain pJexpress- pJexpress401 TAF-CNP inclusion Formicacid Pro-CNP BL21; TAF-CNP bodies (Asp-Pro) BL21(DE3) pJexpress-pJexpress404 KSI-CNP(M/N) CNBr (Met- CNP(M/N) BL21 KSI- inclusion bodiesX) CNP(M/N) pET-31b- pET-31b KSI-CNP inclusion Formic acid Pro-CNPBL21(DE3) KSI-CNP bodies (Asp-Pro) pET-32a- pET-32a Trx-CNP fusionFormic acid Pro-CNP BL21(DE3) Trx-CNP protein (soluble) (Asp-Pro) pMAL-pMAL-c2X MBP-CNP fusion Formic acid Pro-CNP BL21(DE3) CNP protein(soluble) (Asp-Pro) CNP: GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC└Gly-CNP37 (SEQ ID NO: 75); TAF: human transcription factor TAF12; KSI:ketosteroid isomerase; MBP: maltose-binding protein; Trx: thioredoxinExpression of CNP Fusion Proteins in E. coli

CNP fusion protein expression plasmids were transformed into E. coliBL21 or BL21(DE3). Transformed cells were plated on LB plates containing100 ug/ml carbeniciline or 50 ug/ml kanamycin and incubated overnight at37° C. One single colony was picked and cultured in 4 ml LB mediumcontaining 100 ug/ml of carbeniciline or 50 ug/ml kanamycin at 37° C.with shaking. When an OD₆₀₀ of bacterial culture reached 0.6, 1 mMisopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the cell mediaand the media was incubated at 37° C. for 3 hours with shaking. For cellharvest, bacterial cells were centrifuged at 4000 rpm for 10 minutes andthe cell pellets were stored at −80° C. Cell pellets were lysed withB-PER II Bacterial Extraction Reagent (PIERCE, 0.4 ml per 4 ml ofbacterial culture) and Benzonase Nuclease (Novagen, 0.025 U/ml) at roomtemperature for 10 minutes. Bacterial crude extract was reserved andcentrifuged to obtain supernatant. Supernatant and crude extract wereassayed for CNP fusion protein expression and solubility by SDS-PAGE andWestern Blot.

Detection of CNP Fusion Protein Expression with SDS-PAGE and WesternBlot

Ten uL of cell lysates or soluble supernatants was run on sodium dodecylsulfate-polyacrylimide gel electrophoresis (SDS-PAGE) (Invitrogen,Carlsbad, Calif., NuPAGE 4-12% Bis-tris Gel, MES SDS buffer). The gelwas stained using 20 ml Imperial Protein Stain (Thermo Fisher, Rockford,Ill.) at room temperature for 1 hour and de-stained with water. ForWestern blot, the protein was transferred to membrane with Gel blot(Invitrogen). The membrane was blocked in TBS buffer with 5% milk atroom temperature for 1 hour. Rabbit anti-CNP22 antibody (1:2500dilution) (Bachem, Torrance, Calif.) was added to the membrane, whichwas then incubated at room temperature with shaking for 2 hours, andthen the membrane was washed 3 times with TBS buffer.

Alkaline phosphate (AP) conjugated anti-rabbit IgG (1:5000 dilution) wasadded to the membrane, which was then incubated at room temperature withshaking for 1 hour, and then the membrane was washed 3 times with TBSbuffer. Ten ml WESTERN BLUE®Stabilized Substrate (Promega, Madison,Wis.) was added to the membrane, which was then incubated at roomtemperature with shaking for 1 to 5 min, and then the membrane waswashed with TBS buffer to remove excess stain.

Expression of TAF-CNP Fusion Protein in E. coli BL21

Cells (E. coli strain BL21) expressing TAF-CNP fusion protein wereobtained from glycerol stock stored at −80° C. and were grown in 4 ml LBmedium containing 50 ug/ml of kanamycin at 37° C. overnight with shaking(250 rpm). Four ml of overnight grown cell culture was transferred to200 ml LB medium containing 50 ug/ml of kanamycin and was grown at 37°C. with shaking (250 rpm). When the OD₆₀₀ reached 0.6, IPTG was thenadded to a final concentration of 1 mM to induce protein expression at37° C. with shaking (250 rpm) for 3 hours. Cells were then spun down at3000 rpm for 10 minutes and the resulting cell pellet was frozen at −80°C.

Purification of TAF-CNP Inclusion Bodies and Formic Acid Cleavage

The cell pellet (from 200 ml culture) was resuspended in 25 ml of B-PERII buffer (PIERCE), the pellet was sonicated for 10 minutes (50%, 1second, pause 2 seconds) on ice, centrifuged at 12000 rpm for 20 minutesat 4° C., and then the pellet was resuspended in 25 ml 20× diluted B-PERII buffer. This was repeated until the supernatant became clear (3-5times). One mL of resuspended TAF-CNP inclusion bodies was transferredto a 1.5 ml tube and centrifuged at 14000 rpm for 15 min. Thesupernatant was discarded and the pellet was dissolved with 10 ul of 88%formic acid, and then 490 ul of Millipore filtered water was addedimmediately. The pellet was mixed well by vortex and incubated at 55° C.for 20 to 24 hours (70° C./6 hours are alternative conditions). Theproducts of the formic acid cleavage were assayed by SDS-PAGE and LC/MS(C4RP).

LC/MS Sample Preparation

Inclusion bodies were isolated from about 8 mL of culture (about 1.5 OD)and the pellet was solubilized in 10 uL nt formate. Resolubilized pelletwas immediately diluted to 2% or 10% final formate concentration (0.5mL) and incubated at 55° C. for 21 hours (pH 2) (cloudiness was moreevident in the 2% formate sample the next day). Both samples werecentrifuged at 15000 rpm for 2 minutes. Twenty uL of supernatant wasinjected into an LC/MS (C4 RP) apparatus.

Results

CNP Fusion Proteins were Expressed in E. coli

All CNP fusion proteins were expressed in E. coli induced with 1 mM IPTGat 37° C. for 3 hours (FIG. 1). The constructs pJexpress-TAF-CNP,pJexpress-KSI-CNP(M/N) and pET-31b-KSI-CNP were expressed as inclusionbodies, while the constructs pET-32a-Trx-CNP and pMAL-CNP were expressedas soluble fusion proteins. Western blot using anti-CNP22 antibodyconfirmed the expression of the CNP fusion proteins (FIG. 1).

CNP was Produced from TAF-CNP Inclusion Bodies by Formic Acid Cleavage

TAF-CNP inclusion bodies were partially purified and treated with 2%formic acid at 55° C. for 20 to 24 hours (70° C./6 hours are alternativeconditions). The majority of TAF-CNP was cleaved and one extra bandhaving similar size as Gly-CNP37 peptide appeared on SDS-PAGE (FIG. 2).The cleaved sample was further analyzed by LC/MS (C4 RP). The LC/MSresults showed that CNP was released in soluble form from TAF-CNPinclusion bodies after formic acid cleavage. LC/MS analysis indicatedthat formic acid cleavage of the CNP fusion proteins resulted information of cyclized Pro-Gly-CNP37 (MW=4102). Calculation of proteinamounts based on analysis suggested that about 60 ug of formicacid-generated CNP was produced from 8 mL of very low OD culture. From asmall scale (e.g., about 8 mL) of low OD (e.g., 1.2 OD) cell culture,approximately 8 ug/ml CNP was produced, while fermentation of a largerscale (e.g., about 8 L) of higher OD (e.g., 380D) cell culture canproduce approximately 1 mg/ml CNP.

Conclusion

Five expression constructs were generated to express CNP fusionproteins. Expression of all five constructs produced soluble (Trx andMBP) or insoluble (TAF and KSI) CNP fusion proteins. Approximately 1mg/ml soluble CNP can be produced from TAF-CNP inclusion bodies by asimple formic acid cleavage procedure.

Example 2B Production of Additional CNP Variants in E. coli

Recombinant production of CNP variants was carried out as described inExample 2A. In this example, additional CNP constructs were generatedwith a QuikChange II XL site-directed mutagenesis kit (Stratagene) orwere synthesized by DNA2.0. Additional CNP constructs and expressionvectors are listed in Table 7.

TABLE 7 Expression Chemical Final E. coli Construct Vector Productcleavage product strain pJexpress- pJexpress401 TAF(C/A)-Pro- FormicPro- BL21(DE3) TAF(C/A)-Pro- CNP38 inclusion acid CNP38 CNP38 bodies(Asp-Pro) pJexpress- pJexpress401 TAF(4D/4E)-Pro- Formic Pro- BL21(DE3)TAF(4D/4E)- CNP38 inclusion acid CNP38 Pro-CNP38 bodies (Asp-Pro)pJexpress-TAF pJexpress401 TAF(C/A&4D/4E)- Formic Pro- BL21(DE3)(C/A&4D/4E)- Pro-CNP38 inclusion acid CNP38 Pro-CNP38 bodies (Asp-Pro)pJexpress-TAF pJexpress401 TAF(C/A&10D/10E)- Formic Pro- BL21(DE3)(C/A&10D/10E)- Pro-CNP38 inclusion acid CNP38 Pro-CNP38 bodies (Asp-Pro)pJexpress-TAF- pJexpress401 TAF-NL-(C/A & Formic Pro- BL21(DE3)NL-(C/A & 6D/6E)-Pro-CNP38 acid CNP38 6D/6E)-Pro- inclusion bodies(Asp-Pro) CNP38 pJexpress-BMP- pJexpress401 BMP-Pro-CNP38 Formic Pro-BL21; Pro-CNP38 inclusion bodies acid CNP38 BL21(DE3) (Asp-Pro)pJexpress-TAF- pJexpress401 TAF-Pro-CNP37 Formic Pro- BL21; Pro-CNP37inclusion bodies acid CNP37 BL21(DE3) (Asp-Pro) pJexpress-BMP-pJexpress401 BMP-Pro-CNP37 Formic Pro- BL21; Pro-CNP37 inclusion bodiesacid CNP37 BL21(DE3) (Asp-Pro) pJexpress-TAF pJexpress401TAF-Pro-HSA-CNP Formic Pro- BL21 Pro-HSA-CNP inclusion bodies acid HSA-(Asp-Pro) CNP pJexpress-TAF pJexpress401 TAF inclusion bodies N/A N/ABL21(DE3) pJexpress-BMP pJexpress401 BMP inclusion bodies N/A N/ABL21(DE3) pJexpress- pJexpress401 TAF(C/A) inclusion N/A N/A BL21(DE3)TAF(C/A) bodies pJexpress- pJexpress401 TAF(4D/4E) N/A N/A BL21(DE3)TAF(4D/4E) inclusion bodies pJexpress-TAF pJexpress401 TAF(C/A&4D/4E)N/A N/A BL21(DE3) (C/A&4D/4E) inclusion bodies pJexpress-TAFpJexpress401 TAF(C/A&10D/10E) N/A N/A BL21(DE3) (C/A&10D/10E)inclusion bodies pJexpress-TAF- pJexpress401 TAF-NL- N/A N/A BL21(DE3)NL-(C/A & (C/A&6D/6E) 6D/6E) inclusion bodies pJexpress-TAF-pJexpress401 TAF-Pro-CNP53 Formic Pro- BL21(DE3) Pro-CNP53inclusion bodies acid CNP53 (Asp-Pro) pJexpress-TAF- pJexpress401TAF-CNP34 Formic CNP34 BL21(DE3) CNP34 inclusion bodies acid (Asp-Pro)CNP38: GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC [Gly-CNP37 (SEQ ID NO:75)]; Pro-CNP38: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC [Pro-Gly-CNP37](SEQ ID NO: 145); CNP37: QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ IDNO: 60); HSA-CNP: GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC [HSA-CNP27 (SEQID NO: 144)]; Pro-CNP53:PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIG-SMSGLGC (SEQ ID NO:185); CNP34: PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 163);TAF-Pro-CNP38:MVLTKKKLQDLVREVCPNEQLDEDVEEMLLQIADDFIESVVTAA-CQLARHRKSSTLEVKDVQLHLERQWNMWIMGSSHHHHHHSSGLVPRGSHT-GDDDDKHMDPGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO: 196); TAF: human transcription factor TAF12 histone folddomain (HFD) and linker from pET-15b vector; TAF-NL: TAF12 HFD without alinker; TAF12 HFD:VLTKKKLQDLVREVCPNEQLDEDVEEMLLQIADDFIESVVTAACQLA-RHRKSSTLEVKDVQLHLERQWNMWI(SEQ ID NO: 197); pET-15b linker: MGSSHHHHHHSSGLVPRGSHTGDDDDKHMD (SEQ IDNO: 195); TAF(C/A): cysteine in TAF is changed to alanine; TAF(D/E):aspartic acid in TAF is changed to glutamic acid (number indicates whichamino acid residue is changed); TAF(C/A & D/E): cysteine and asparticacid in TAF are changed to alanine and glutamic acid, respectively; BMP:bone morphogenetic protein 2 with seven C/A (cysteine to alanine)mutations; KSI: ketosteroid isomerase; MBP: maltose-binding protein;TRX: thioredoxinResults

All CNP fusion proteins were expressed in E. coli induced with 1 mM IPTGat 37° C. for 3 hours. Constructs pJexpress-BMP-Pro-CNP38,pJexpress-TAF-Pro-CNP37, pJexpress-BMP-Pro-CNP37, pJexpress-Pro-HSA-CNP,pJexpress-TAF and pJexpress-BMP were expressed as inclusion bodies. AWestern Blot with anti-CNP antibody was used to confirm the expression(FIG. 3).

Pro-Gly-CNP37 (“Pro-CNP38”) was Produced from TAF-Pro-CNP38 InclusionBodies by Formic Acid Cleavage

Formic acid is used as a denaturant for inclusion body proteins and canspecifically cleave the peptide bond between Asp and Pro under optimizedconditions. TAF-Pro-CNP inclusion bodies were partially purified asdescribed above and treated with 50% formic acid at 25° C., 37° C., 42°C. and 55° C. for 24 hours. Most of the TAF-Pro-CNP38 was cleaved andone extra band with a similar size as the Gly-CNP37 (“CNP38”) peptideshowed on SDS-PAGE from the 37° C., 42° C. and 55° C. cleavages (FIG.4A). The cleavage reactions at 37° C. and 55° C. were neutralized with10 M NaOH and centrifuged at 14,000 rpm for 15 minutes. The un-cleavedTAF-Pro-CNP38, TAF and other inclusion bodies precipitated in thepellets. The supernatants contained soluble Pro-CNP38 and were furtheranalyzed by LC/MS. The LC/MS result showed the supernatants contained amixture of nonspecific cleaved peptides generated by excess acidichydrolysis.

When TAF-Pro-CNP inclusion bodies were treated with 2% and 10% formicacid at 55° C. for 20 hours, the majority of TAF-Pro-CNP38 was cleavedand one extra band having a similar size as Gly-CNP37 was observed onSDS-PAGE (FIG. 4B). The cleaved sample was further analyzed by LC/MS.The LC/MS analysis showed that correct Pro-CNP38 was released in solubleform from TAF-Pro-CNP38 inclusion bodies after formic acid cleavage. Theyields of Pro-CNP38 from 2% and 10% formic acid cleavages were similar(FIG. 4C).

Formic Acid Cleavage and Neutralization for Pro-Gly-CNP37 (“Pro-CNP38”)Production and Purification

Formic acid can dissolve and cleave TAF-Pro-CNP38, BMP-Pro-CNP38 andother inclusion bodies. The pH neutralization results in precipitationof insoluble contaminating proteins/peptides (un-cleaved TAF-Pro-CNP38and BMP-Pro-CNP38, TAF, BMP and others). The soluble Pro-CNP38 stays inthe supernatant after centrifugation. TAF-Pro-CNP38 and BMP-Pro-CNP38were cleaved in 2% formic acid at 55° C. or 70° C. for 24 hours. Thecleavage reactions were neutralized with 1:1 ratio of 0.5 M Tris bufferand centrifuged at 14,000 rpm for 15 minutes. Results showed that thesupernatants contained almost pure peptides and there was no observedrecovery loss of Pro-CNP38 upon neutralization. This is a simple andefficient step for Pro-CNP38 purification.

Analysis of Varying Conditions for Formic Acid Cleavage of TAF-Pro-CNP38Inclusion Bodies for Pro-Gly-CNP37 (“Pro-CNP38”) Production

Formic acid can specifically cleave the peptide bond between Asp and Prounder optimized conditions. Non-specific cleavage of peptide bondsbetween Asp and any other amino acids or even non-specific cleavage ofany peptide bond may occur if the formic acid cleavage conditions arenot optimized. TAF-Pro-CNP38 inclusion bodies were cleaved with 2%formic acid at 42° C., 55° C. or 70° C. for 6, 24 or 48 hours. FIG. 5Ashows that TAF-Pro-CNP38 was cleaved completely at 70° C. for 24 hoursor at 55° C. for 48 hours. The 70° C. cleavage could be completed within17 hours (FIG. 5B). Although the 70° C./24 h cleavage gave the highestyield, the non-specific cleavage products (for example, the peptide withmolecular weight of 3142 generated from Pro-CNP38 by cleaving betweenpeptide bond Asp and Arg) increased dramatically (FIG. 5C).

The yield and purity of Pro-CNP38 production were improved whenTAF-Pro-CNP38 inclusion bodies were purified or treated with B-PER IIbuffer before formic acid cleavage. Because B-PER II buffer containsdetergent octylthioglucoside and is relatively expensive, other commonlyused detergents or buffers were tested for large-scale Pro-CNP38production. TAF-Pro-CNP38 inclusion bodies were re-suspended indifferent detergents (Octylsucrose; Triton x-100; Tween-20; NP-40;CA-630) or buffers (B-PER II; B-PER II 1/20 dilution; B-PER; B-PERphosphate buffer; 25 mM tris, 150 mM NaCl, pH 7.9; 25 mM tris, pH 7.5;filtered water; PBS) and incubated at room temperature (RT) for 24hours. All detergents were 1% in 25 mM tris buffer, pH 7.5. Afterincubation in detergent or buffer, TAF-Pro-CNP38 inclusion bodies werecleaved by 2% formic acid at 55° C. for 22 hours. Results showed thatBPER II continued to exhibit good yield, and positive results were alsoobtained with CA630 and Triton X-100.

Pro-Gly-CNP37 (“Pro-CNP38”) Proteolytic Cleavage Products

One unidentified protease, possibly a membrane associated protease,cleaved the Pro-CNP38 peptide (produced from BL21 strain, MW 4102) intotwo peptides during Pro-CNP38 purification, resulting in peptidesPGQEHPNAR (MW 1004) (SEQ ID NO: 198) and KYKGANKKGLSKGCFGLKLDRIGSMSGLGC(MW 3115) (SEQ ID NO: 199). Not to be bound by theory, one possiblereason why detergents may improve the yield and purity of Pro-CNP38 isthat detergents can remove most, but possibly not all, of theunidentified protease. High temperature, basic pH and EDTA were testedto identify if these agents could inhibit the protease cleavage.TAF-Pro-CNP38 inclusion bodies were incubated at RT or 120° C. for 2 hrsand centrifuged at 14000 rpm for 15 min. The pellets were re-suspendedin 2% formic acid and incubated at 55° C. or 70° C. for 18 hours. A 1:1ratio of 0.5 M Tris was added to neutralize the cleavage. Theneutralized samples were centrifuged at 14000 rpm for 5 min and thesupernatants were left at RT for 6 hrs or 22 hrs with or without 10 mMEDTA, pH 10. The proteolytic cleavage was assayed by LC/MS (Table 8).

TABLE 8 LC/MS result of Pro-CNP38 proteolytic cleavage A210 Protein PeakConc. Percent Percent Sample Area (mg/mL) MW4102 MW3115 1 H 55 C. 9420.04 * 91.8 8.2 2 H 70 0 2878 0.11 * 79.2 20.7 3 H 70 6H 2675 0.10 *80.6 19.4 4 H 70 24 2741 0.11 * 79.2 20.7 5 H 70 EDTA 2385 0.09 * 80.819.2 6 H 70 pH 10 1917 0.07 * 81.2 18.8 7 55 C. 1291 0.05 61.1 38.9 8 70C. 0 4533 0.18 96.8 3.2 9 70 C. 6H 4120 0.16 97.5 2.5 10 70 C. 24 41080.16 96.5 3.5 11 70 C. EDTA 4336 0.17 96.9 3.1 12 70 C. pH 10 3425 0.1397.5 2.4

All samples (8-12) cleaved at 70° C. showed limited proteolytic cleavage(less than 4% cleavage of Pro-CNP38). Almost 40% of Pro-CNP38 wascleaved by protease when cleavage was carried out at 55° C. (sample 7).Basic pH and EDTA did not influence the non-specific proteolyticcleavage. High temperature (120° C. for 2 hrs) non-specifically cleavedPro-CNP38.

It should be noted that the BL21(DE3) strain from Stratagene does nothave the unidentified protease.

Production of Pro-Gly-CNP37 (“Pro-CNP38”) and Other CNP Variants fromDifferent Constructs:

Pro-CNP38 can be produced in large scale in E. coli by means ofoverexpression of a TAF-Pro-CNP38 fusion protein as inclusion bodiesfollowed by formic acid cleavage of the fusion protein. Following themethods described herein, other TAF-CNP fusion proteins (TAF-CNP34 andTAF-Pro-CNP53) were expressed as inclusion bodies and then cleaved withformic acid to generate the CNP variants CNP34 and Pro-CNP53. FIG. 6depicts the expression of TAF-CNP34, FIG. 7 the expression ofTAF-Pro-CNP53, FIG. 8 the products of formic acid cleavage of TAF-CNP34and TAF-Pro-CNP53, FIG. 9 the peak for CNP-34 in an LC/MS chromatogram,and FIG. 10 the peak for Pro-CNP53 in an LC/MS chromatogram.

Use of formic acid may result in unspecific cleavage(s) at peptidebond(s) other than the targeted Asp-Pro bond. To improve the purity andoverall titer of the desired formic acid cleavage product, differentresidues of aspartic acid in TAF12 or fragments thereof were changed toglutamic acid. Moreover, one or more cysteine residues in TAF12 orfragments thereof were changed to alanine to prevent unspecificdisulfide bond formation. All TAF-Pro-CNP38 fusion proteins having suchmutations in TAF12 were expressed as inclusion bodies and cleaved withformic acid to produce Pro-CNP38. FIG. 7 shows the expression ofTAF-NL-(C/A & 6D/6E)-Pro-CNP38 and TAF(C/A & 10D/10E)-Pro-CNP38, FIG. 11the expression of TAF(C/A & 4D/4E)-Pro-CNP38 and TAF(4D/4E)-Pro-CNP38,FIG. 12 the products of formic acid cleavage of TAF(4D/4E)-Pro-CNP38 andTAF(C/A & 4D/4E)-Pro-CNP38, and FIG. 13 the products of formic acidcleavage of TAF-NL-(C/A & 6D/6E)-Pro-CNP38 and TAF(C/A &10D/10E)-Pro-CNP38. Table 9 summarizes the purity (prior topurification) and titer of Pro-CNP38 obtained from the variousTAF-Pro-CNP38 constructs.

TABLE 9 Construct Purity Titer (ug/mL) pJexpress-TAF-Pro-CNP38 32% 44pJexpress-TAF(C/A)-Pro-CNP38 41% 50 pJexpress-TAF(4D/4E)-Pro-CNP38 36%52 pJexpress-TAF(C/A & 4D/4E)-Pro-CNP38 42% 58 pJexpress-TAF(C/A &10D/10E)-Pro-CNP38* 32% 26 pJexpress-TAF-NL-(C/A & 6D/6E)-Pro-CNP38 50%55 *Cells with pJexpress-TAF(C/A&10D/10E)-Pro-CNP38 growed more slowlyand the final cell density (OD₆₀₀) was lower compared to otherTAF-Pro-CNP38 constructs.Large-scale Production of Pro-Gly-CNP37 (“Pro-CNP38”) by Fermentationand Formic Acid Cleavage

BL21(DE3) cells comprising the pJexpress-TAF-CNP construct were grown ina 10 liter fermenter at 37° C. for about 16-17 hours until OD₆₀₀ reached64. The cells were then grown/cultured in the presence of 1 mM IPTG atabout 35-37° C., to induce expression of the TAF-Pro-CNP38 fusionprotein, for about 7-8 hours until OD₆₀₀ reached 160. The fermentationproduced a titer of 9 g/L TAF-Pro-CNP38. FIG. 14 displays a Western blotof the TAF-Pro-CNP38 fusion protein produced in the fermentation.

A cell pellet recovered from 750 mL cell culture from the 10 Lfermentation was resuspended in phosphate-buffered saline, pH 7.4 (PBS)and lysed by three passes through a pressure homogenizer (10,000 bar).The resulting lysate was centrifuged at 6,500 g for 10 minutes and thesupernatant was discarded. The pellet fraction containing insolubleTAF-Pro-CNP38 fusion protein inclusion bodies was resuspended in 500 mLPBS with a rotostator. The suspension was centrifuged at 6,500 g for 10minutes and the supernatant was discarded. The resulting inclusion bodypellet was resuspended in 500 mL water with a rotostator and incubatedat 55° C. for 30 minutes. 250 mL 6% formic acid was added to the warmedinclusion body suspension for a final concentration of 2% formic acidand incubated at 55° C. for 20-24 hours. 50 mL of 400 mM Na₂HPO₄ wasadded after 20-24 hours to begin the neutralization of the formic acidcleavage reaction, and the resulting mixture was titrated with 50% w/vNaOH to pH 6.9-7.4 and left at room temperature for 30 minutes. Uponneutralization, a heavy precipitate formed and was removed bycentrifugation at 6,500 g for 10 minutes. The supernatant was retainedand contained on average 1.3 g/L culture of 80% pure Pro-CNP38. Themajority of the E. coli and TAF-related proteins and peptides remainedin the pellet.

The soluble Pro-CNP38 resulting from the formic acid cleavage andneutralized supernatant was 80% pure and contained a mixture of linearand cyclized Pro-CNP38 peptides along with other product-relatedimpurities. The pH neutral supernatant containing Pro-CNP38 wassterile-filtered. Further purification by anion-exchange chromatographyusing a Fractogel TMAE Hi-CAP column (EMD Biosciences) to remove DNA,endotoxins, and peptide contaminants, which should bind to the column,was performed at pH 7-7.4. The flow-through fraction contained partiallycyclized Pro-CNP38. Cupric sulfate was added to the flow-throughfraction to a final concentration of 10 uM and incubated at roomtemperature for 1 hour. The addition of Cu⁺⁺ at neutral pH catalyzed theoxidation of free cysteine sulfhydryl groups on the peptide to form anintramolecular disulfide bond, resulting in 100% cyclized Pro-CNP38 andno detectable linear peptide. The conductivity of the solution wasadjusted to <15 mS/cm by the addition of water. Cation-exchangechromatography using an SP-Sepharose column (GE Healthcare) and a sodiumphosphate buffer (pH 7) was then performed to remove any remaining DNAand endotoxins in the flow-through and further purify Pro-CNP38 to about95-96% purity with <0.5% non-product related impurities. FIG. 15 is anSDS-PAGE of eluate fractions from the SP-Sepharose column; the highestconcentrations of Pro-CNP38 were found in fractions 22 to 30.Reverse-phase HPLC/MS analysis of non-pooled fraction 24 indicated thepresence of 90% Pro-CNP38, 5% Pro-CNP38 with an oxidized methionineresidue, 3% Pro-CNP38 cleaved at the Gly-Cys bond to form CNP-17, and1.6% Pro-CNP38 cleaved at the Asp-Arg bond in the cyclic domain. Theyield of pure Pro-CNP38 peptide upon final purification was 0.9 g/L cellculture (36% total recovery).

Pro-CNP38 collected from five separate purifications was pooled forformulation. The pooled product contained 93.5% Pro-CNP38, 3.3%Pro-CNP38 with an oxidized methionine residue, 1.3% deamidatedPro-CNP38, and 1% Pro-CNP38 cleaved at the Gly-Cys bond to form CNP-17.Samples were diluted with 50 mM sodium phosphate (pH 7) to aconductivity of 10 mS/cm and loaded onto a CM-Sepharose column(GE-Healthcare) for concentration and buffer exchange. The weakcation-exchange property of the CM-Sepharose resin allows peptides todissociate from the column with weak acid solutions rather than typicalsalt gradients. Acid concentrations required for dissociation ofPro-CNP38 from the CM-Sepharose column depended upon column loading. At50 mg Pro-CNP38 per mL of resin, 10 mM HCl was sufficient for Pro-CNP38elution. At 9 mg Pro-CNP38 per mL of resin, 50 mM HCl was required forelution. When the loading was 9 mg Pro-CNP38 per mL of resin, Pro-CNP38eluted in less than one column volume, which significantly concentratedthe peptide. The eluate fraction contained 20.3 mg/mL of 95% purePro-CNP38, along with 3% Pro-CNP38 with an oxidized methionine residue,1% deamidated Pro-CNP38, and <1% Pro-CNP38 cleaved at the Gly-Cys bondto form CNP-17. The concentrated solution of Pro-CNP38 in weak acid issuitable for dilution into appropriate buffers for either liquid orlyophilized formulations.

Example 3 Cleavage of CNP Variants by Neutral Endopeptidase In Vitro

To determine the effects of amino acid substitutions, amino acidextensions, backbone modifications, side chain modifications andPEGylation on the susceptibility of CNP variants to neutralendopeptidase (NEP) cleavage, peptide cleavage assays were carried outusing an in vitro assay that monitored disappearance of the non-cleavedCNP variant.

Recombinant human NEP (1 ug/mL final concentration) was added to 100 uMCNP variant diluted in 0.1 M Tris, pH 7. The reaction mixture wasincubated at 37° C. for various periods of time, and the reaction wasquenched with EDTA (10 mM final) followed by heat denaturation. Thereaction mixture was reduced and then the reaction products wereanalyzed using HPLC and mass spectroscopy. The half-life of the CNPvariant was calculated based on the disappearance of intact CNP variantover time. The results for digested CNP variants were compared to aparallel wtCNP22 digestion and normalized to the results for 100 uMCNP22 digested by 1 mg/mL NEP (t_(1/2)=80 min).

Table 1 lists the half-lives, based on the in vitro NEP cleavage assay,of various CNP variants having backbone or side chain modifications.Removal of three of the six NEP cleavage sites in Analog L neverthelessresulted in a substantially shorter half-life. Of tested CNP variants,the greatest resistance to NEP cleavage was exhibited by Analog N, whichcontains the D-enantiomer of all 22 amino acids of CNP22, and by AnalogM, which has an N-methylated amide bond at both Leu9 and Leu11. However,both Analogs N and M failed to stimulate production of cGMP (see below).

Comparing the half-lives of Analogs A, B, E, F, G and H to one another,half-lives were determined to be about 1.5- to about 2.5-fold longer forAnalogs E and G compared to those for Analogs A, B, F and H. All ofthese six analogs showed resistance or improved resistance to cleavageat the Cys6-Phe7 bond relative to wtCNP22 (data not shown). The rankorder of analog resistance to NEP at 1 ug/ml, based on half-life, isAnalog G (3-Cl-Phe)≧Analog E (D-Phe)>Analog H (“beta-2 Phe”), Analog B(N-Me-Phe), and Analog F (t-Bu-Gly)=wtCNP22>Analog A (Cys-CH₂—NH).Analogs E and G have about 1.5 times longer half-life in comparison towtCNP22. Besides resistance to cleavage of the Cys6-Phe7 bond, AnalogsB, E, F, G and H also exhibited resistance to cleavage of the Gly8-Leu9bond in the presence of 1 ug/mL NEP (data not shown). These resultsindicate that CNP variants having backbone or side chain modificationsbetween Cys6 and Gly8 can be resistant to NEP cleavage of the Cys6-Phe7bond and/or Gly8-Leu9 bond, but do not necessarily have improved overallresistance to NEP or a longer half-life than CNP22. The results seem tobe contrary to reports in the literature that NEP first cleaves at theCys6-Phe7 bond of CNP22 and then elsewhere.

TABLE 1 cGMP Response rel. NEP Backbone and Side Chain Modifications to1 uM CNP22¹ Cleavage Analog Natriuretic Peptide 10 nM 1 uM (t_(1/2),min) CNP22 (SEQ ID NO: 1) 46 ± 10 100 ± 13  80² N D-CNP22 (all D-aminoacids) (SEQ ID NO: 115) 2 1 >>160   A CNP22, C6—CH2—NH (reducedcarbonyl) (SEQ ID 6 66 55 NO: 56) B CNP22, N-methyl-F7 (methylatedamide) (SEQ ID 2 38 80 NO: 57) BD CNP22, N-methyl-L9 (SEQ ID NO: 116) 28 ND BN CNP22, N-methyl-L11 (SEQ ID NO: 117) 10 51 ND BE CNP22,N-methyl-L20 (SEQ ID NO: 118) 2 5 ND M CNP22, N-methyl-L9, N-methyl-L11(SEQ ID NO: 1 11 >>160   94) K CNP22, N-methyl-L9, N-methyl-L20 (SEQ IDNO: 1 1 80 92) L CNP22, N-methyl-L9, N-methyl-L11, N-methyl-L20 18 10 30(SEQ ID NO: 93) J CNP22, C6—CH2—NH, N-methyl-L9, N-methyl-L20 ND ND 50(SEQ ID NO: 91) E CNP22, D-F7 (D-Phe) (SEQ ID NO: 136) 2 6 130  H CNP22,Beta-2-F7 (3-amino-2-phenylpropionyl) 2 2 80 (SEQ ID NO: 57) G CNP22,3-chloro-F7 (SEQ ID NO: 137) 17 93 135  F CNP22, t-butyl-G8 (SEQ ID NO:58) 2 18 80 V CNP22, K4G, 3,4-dichloro-F7 (SEQ ID NO: 119) ND ND 68 XCNP22, K4G, 3-methyl-F7 (SEQ ID NO: 120) ND ND 68 ANP 10 23 ND¹Stimulation of cGMP production in NIH3T3 cells by natriuretic peptiderelative to cGMP production in the presence of 1 uM CNP22 ²CNP22 NEPresistance t_(1/2) averaged 80 min. Due to variations in NEP catalyticactivity between experiments, all CNP22 t_(1/2) digestions werenormalized to 80 min and the difference coefficient was used tocalculate analog t_(1/2) in each experiment to obtain an adjustedt_(1/2). ND = Not Determined

Table 2 lists the half-lives, based on the in vitro NEP cleavage assay,of various CNP variants having substitutions with natural and/orunnatural amino acids. Of tested variants, the greatest resistance toNEP cleavage was shown by Analog BK, which has K4R and G15Ssubstitutions, and Analog BJ, which has K4R and G15N substitutions.

TABLE 2 cGMP Response relative NEP Specificity Mutations to 1 uM CNP22¹Cleavage Analog Natriuretic Peptide 10 nM 1uM (t_(1/2), min) CNP22 (SEQID NO: 1)  46 ± 10 100 ± 13  80 AH CNP22, K4R (SEQ ID NO: 35) 59 121 80BP CNP22, K4R, G5S (SEQ ID NO: 121) 45 ND ND BO CNP22, K4R, G5R (SEQ IDNO: 122) 18 80 ND P CNP22, K4G (SEQ ID NO: 123) ND ND 68 Z CNP22, K4R,F7Y (SEQ ID NO: 95) 2 18 ND AB CNP22, K4R, G8S (SEQ ID NO: 97)  26 ± 2686 ± 17 ND AA CNP22, K4R, G8V (SEQ ID NO: 96) 3 25 ND AC CNP22, K4R, G8T(SEQ ID NO: 98) 11 ± 2 66 ± 16 80 AD CNP22, K4R, L9T (SEQ ID NO: 99) 468 ND BH CNP22, K4R, K10R (SEQ ID NO: 112) 12 80 ND BF CNP22, K4R,K10Cit (SEQ ID NO: 110) 6 33 ND BG CNP22, K4R, K10Q (SEQ ID NO: 111) 945 ND BY CNP22, K4R, K10S (SEQ ID NO: 124) 16 53 ND BK CNP22, K4R, G15S(SEQ ID NO: 114) 13 ± 1 71 ± 11 ≧160 BJ CNP22, K4R, G15N (SEQ ID NO:113) 4 41 150 AE CNP22, K4R, G15R (SEQ ID NO: 100) 0.3 0.3 ND AF CNP22,K4R, G15Cit (SEQ ID NO: 101) 1.4 2 ND BZ CNP22, K4R, S16Q (SEQ ID NO:125) 42 116 ND BX CNP22, K4R, M17N (SEQ ID NO: 126) 40 ± 2 103 ± 17  NDAG CNP22, K4R, M17V (SEQ ID NO: 102) 10 65 ND BQ CNP22, K4R, G19S (SEQID NO: 127) 21 63 ND BR CNP22, K4R, G19R (SEQ ID NO: 128) 22 ± 6 84 ± 10ND AJ CNP22, K4R, L20V (SEQ ID NO: 103) 0.2 8 ND AK CNP22, K4R,L20t-butyl-Ala 1 21 ND (SEQ ID NO: 104) AT CNP22, G1E, K4E (SEQ ID NO:105) 11 54 60 BS CNP22, K4R, L20R (SEQ ID NO: 129) 11 8 ND BT CNP22,K4R, G21S (SEQ ID NO: 130) 7 39 ND BU CNP22, K4R, G21T (SEQ ID NO: 131)6 21 ND BW CNP22, K4R, G21R (SEQ ID NO: 132) 20 21 ND ANP 10 23 ND¹Stimulation of cGMP production in NIH3T3 cells by natriuretic peptiderelative to cGMP production in the presence of 1 uM CNP22 ²CNP22 NEPresistance t_(1/2) averaged 80 min. Due to variations in NEP catalyticactivity between experiments, all CNP22 t_(1/2) digestions werenormalized to 80 min and the difference coefficient was used tocalculate analog t_(1/2) in each experiment to obtain an adjustedt_(1/2). ND = Not Determined

Table 3 lists the half-lives, based on the in vitro NEP cleavage assay,of CNP variants having N-terminal and/or C-terminal modifications,including amino acid extensions. Of the analogs tested, Analogs AZ, CC,CF, BL, CS, CK and CL, Pro-Gly-CNP37 and HSA-CNP27 were most resistantto NEP degradation.

TABLE 3 cGMP Response rel. NEP N- and C-Terminal Modificationsto 1 uM CNP22¹ Cleavage Natriuretic Peptide 10 nM 1 uM (t_(1/2), min)CNP22 46 ± 10 100 ± 13 80 BCPentanoic acid (N-term.)-CNP22, G1E (SEQ ID NO: 109) ND ND ND BBHeptanoic acid (N-term.)-CNP22, G1E (SEQ ID NO: 108) 32 ± 4  84 ± 1945-65 AV Pentanoic acid (N-term.)-CNP22, G1E, K4E (SEQ ID NO: ND ND 120106) AW Heptanoic acid (N-term.)-CNP22, G1E, K4E (SEQ ID NO: ND ND <20107) AX CNP17 (delta N-term) (SEQ ID NO: 2) 18 69 NDR-CNP22 (SEQ ID NO: 40) ND ND ND AZ R-CNP22, K4R (SEQ ID NO: 41) 54 ± 11106 ± 15  ≧160 ER-CNP22 (SEQ ID NO: 38) ND ND ND BAER-CNP22, K4R (SEQ ID NO: 39) 38 ± 10 113 ± 10  90GANRR-CNP22 (SEQ ID NO: 65) ND ND ND AY GANRR-CNP22, K4R (SEQ ID NO: 36)59 ± 9  105 ± 20  65 GANQQ-CNP22 (SEQ ID NO: 64) ND ND ND CHGANQQ-CNP22, K4R (SEQ ID NO: 69) 44 ± 8  95 ± 11 NDGANPR-CNP22 (SEQ ID NO: 66) ND ND ND CI GANPR-CNP22, K4R (SEQ ID NO: 37)50 ± 1  105 ± 12  ND GANSS-CNP22 (SEQ ID NO: 67) ND ND ND CGGANSS-CNP22, K4R (SEQ ID NO: 70) 27 ± 1  88 ± 1  95 CAAAWARLLQEHPNA-CNP22 (SEQ ID NO: 61) 24 76 ND CBAAWARLLQEHPNAR-CNP22 (SEQ ID NO: 62) 36 84 ND CCDLRVDTKSRAAWAR-CNP22 (SEQ ID NO: 63) 34 101 >160 CFGQPREPQVYTLPPS-CNP22 (IgG1(Fc) fragment) 23 ± 9  72 ± 19 >160(SEQ ID NO: 79) PNARKYKGANKK-CNP22 (CNP34) ND ND ND BLQEHPNARKYKGANKK-CNP22 (CNP37) 43 ± 15 97 ± 27 >>160 (SEQ ID NO: 60)PQEHPNARKYKGANKK-CNP22 (Pro-CNP37) ND ND ND CEGERAFKAWAVARLSQ-CNP22 (HSA fragment) 15 87 ND (SEQ ID NO: 81) CYGQHKDDNPNLPRGANPR-CNP22 (HSA fragment) ND ND ND (SEQ ID NO: 80) CQGHHSHEQHPHGANQQ-CNP22 (HRGP fragment) 16 95 ND (SEQ ID NO: 76) CXGHHSHEQHPHGANPR-CNP22 (HRGP fragment) ND ND ND (SEQ ID NO: 78) CSGQEHPNARKYKGANPK-CNP22 (modified CNP37) 19 61 >>160 (SEQ ID NO: 129) CTGQEHPNARKYKGANQK-CNP22 (modified CNP37) 60 121 ND (SEQ ID NO: 130) CUGQEHPNARKYKGANQQ-CNP22 (modified CNP37) 9 57 ND (SEQ ID NO: 131) DBGQEHPNARKYKGANKK-CNP22 (Gly-CNP37) 50 ± 14 98 ± 17 >>160 (SEQ ID NO: 75)PGQEHPNARKYKGANKK-CNP22 (Pro-Gly-CNP37) 49 ± 6  103 ± 17  >>160 CWGQEHPNARKYKGANKP-CNP22 (modified CNP37) ND ND ND (SEQ ID NO: 74) CRGAHHPHEHDTHGANQQ-CNP22 (HRGP fragment) 14 ± 5 77 ± 12 ND(SEQ ID NO: 128) CZ FGIPMDRIGRNPR-CNP22 (osteocrin “NPR-C inhibitor”) NDND ND (SEQ ID NO: 82) DA GKRTGQYKLGSKTGPGPK-CNP22 (FGF2 “heparin- ND NDND binding domain” fragment) (SEQ ID NO: 83) CKGQPREPQVYTGANQQ-CNP22, K4R (IgG1(Fc) fragment) 2 32 ≧160 (SEQ ID NO: 84)CL GVPQVSTSTGANQQ-CNP22, K4R (HSA fragment) 3 35 >160 (SEQ ID NO: 85)GHKSEVAHRFKGANKK-CNP22 (HSA-CNP27) (SEQ ID 51 ± 9  109 ± 15  >>160NO: 144) PGHKSEVAHRFKGANKK-CNP22 (Pro-HSA-CNP27) 32 107 ND CNGQTHSSGTQSGANQQ-CNP22, K4R (fibrinogen) 12 115 ND (SEQ ID NO: 87) CMGQPSSSSQSTGANQQ-CNP22, K4R (fibronectin) ND ND ND (SEQ ID NO: 86) COGSTGQWHSESGANQQ-CNP22, K4R (fibrinogen) 2 33 ND (SEQ ID NO: 88) CPGSSSSSSSSSGANQQ-CNP22, K4R (zinc finger) ND ND ND (SEQ ID NO: 89) CDSPKMVOGSG-CNP17-KVLRRH (“BNP tails”) 25 102 ND (SEQ ID NO: 68) CJRSSCFGGRIDRIGAC (“C-ANP4-23”, ANP-derived) ND ND ND (SEQ ID NO: 133)CNP22, K4R, K10R, N.-term.--N-term. dimer/ 19 44 NDdisuccinimidyl glutarate (SEQ ID NO: 134)CNP22, K4R, K10R, N-term.--N.-term. dimer/Bis-PEO5 19 41 ND(SEQ ID NO: 135) BM CNP53 (SEQ ID NO: 4) 61 101 >>160 ANP 10 23 ND¹Stimulation of cGMP production in N1H3T3 cells by natriuretic peptiderelative to cGMP production in the presence of 1 uM CNP22 ²CNP22 NEPresistance t_(1/2) averaged 80 min. Due to variations in NEP catalyticactivity between experiments, all CNP22 t_(1/2) digestions werenormalized to 80 min and the difference coefficient was used tocalculate analog t_(1/2) in each experiment to obtain an adjustedt_(1/2). ND = Not Determined

Table 4 lists the half-lives, based on the in vitro NEP cleavage assay,of CNP variants conjugated to PEG (or PEO) polymers at the N-terminus.All the PEGylated CNP variants tested and shown in Table 4 displayedresistance or enhanced resistance to NEP cleavage except forPEO12-GANPR-CNP22(K4R), which had the same half-life as wtCNP22.N-terminal PEGylation of CNP22 having a K4G substitution does not seemto confer substantial improvement in NEP resistance. For example,PEG2K-CNP22(K4G) was only slightly more resistant to NEP cleavage thanCNP22 (data not shown), whereas PEG2K-CNP22 had a much longer half-lifein vitro than CNP22.

TABLE 4 cGMP Response rel. to 1 uM NEP N-Terminal PEGylation CNP22¹Cleavage Natriuretic Peptide PEG 10 nM 1uM (t_(1/2), min) CNP22  46 ± 10100 ± 13  80 CNP22 PEG20K 0 15 >>160 CNP22 PEG5K  8 ± 1 20 ± 7  >>160CNP22 PEG2K  6 ± 2 32 ± 4  >>160 CNP22 PEO4-(PEO12)₃ 17 ± 1 52 ±6  >>160 (branched) CNP22 PEO24 (1.2 kDa)  8 ± 5 46 ± 10 >>160 CNP22PEG1K 15 ± 3 68 ± 17 >160 CNP22 PEO12 (0.6 kDa) 12 ± 7 57 ± 18 160 CNP22(PEO12)-Biotin 19 81 140 CNP22, K4G (PEO12)-Biotin 10 27 100 CNP22, K4RPEO24 15 56 ND CNP22, K4R PEO12 13 44 ND CNP-17 PEG2K 5 50 >160 R-CNP22,K4R (SEQ ID NO: 41) PEO24 15 ± 2 75 ± 12 ND R-CNP22, K4R (SEQ ID NO: 41)PEO12 23 ± 2 93 ± 19 ≧160 ER-CNP22, K4R (SEQ ID NO: 39) PEO24  6 ± 2 60± 10 ND ER-CNP22, K4R (SEQ ID NO: 39) PEO12 20 ± 1 92 ± 25 NDGANRR-CNP22, K4R (SEQ ID NO: 36) PEG2K 15 ± 2 45 ± 18 ND GANRR-CNP22,K4R (SEQ ID NO: 36) PEO24 28 ± 9 82 ± 18 >>160 GANRR-CNP22, K4R (SEQ IDNO: 36) PEG1K   15 ± 0.4 56 ± 23 >160 GANRR-CNP22, K4R (SEQ ID NO: 36)PEO12 40 ± 2 99 ± 13 >160 GANQQ-CNP22, K4R (SEQ ID NO: 69) PEO24  16 ±13 73 ± 30 ND GANQQ-CNP22, K4R (SEQ ID NO: 69) PEO12 30 78 NDGANPR-CNP22, K4R (SEQ ID NO: 37) PEO24 ND ND ND GANPR-CNP22, K4R (SEQ IDNO: 37) PEO12 ND ND 80 GANSS-CNP22, K4R (SEQ ID NO: 70) PEO24  8 ± 5 46± 21 ND GANSS-CNP22, K4R (SEQ ID NO: 70) PEO12   8 ± 0.3 52 ± 13 ND¹Stimulation of cGMP production in NIH3T3 cells by natriuretic peptiderelative to cGMP production in the presence of 1 uM CNP22 ²CNP22 NEPresistance t_(1/2) averaged 80 min. Due to variations in NEP catalyticactivity between experiments, all CNP22 t_(1/2) digestions werenormalized to 80 min and the difference coefficient was used tocalculate analog t_(1/2) in each experiment to obtain an adjustedt_(1/2). ND = Not Determined

FIG. 16 shows the NEP resistance profile of five N-terminal PEGylatedconjugates of CNP22. The CNP22 peptides conjugated to PEG (or PEO)polymers of increasing mass exhibited increasing resistance to NEPdegradation. In particular, PEO24-CNP22, PEG2K-CNP22 and PEG5K-CNP22were resistant to NEP degradation over the assay period of 160 minutes.

FIG. 17 displays the NEP resistance profile of CNP variants CNP37(Analog BL), CNP53 and GANRR-CNP22(K4R) (SEQ ID NO: 36) having anN-terminal amino acid extension. As can be clearly seen, both CNP37 andCNP53 were resistant to NEP degradation in this in vitro assay, whereasGANRR-CNP22(K4R) (SEQ ID NO: 36) had the same lability to NEP hydrolysisas CNP22.

FIG. 18 depicts the NEP resistance profile of CNP17 and GANRR-CNP22(K4R)(SEQ ID NO: 36) conjugated to a PEG (or PEO) moiety at the N-terminus.PEGylation of GANRR-CNP22(K4R) (SEQ ID NO: 36) greatly improved the NEPresistance of this CNP variant, with PEO24-GANRR-CNP22(K4R) (SEQ ID NO:36) being completely resistant to NEP cleavage over the assay period of160 minutes. Increasing the mass of the PEO moiety from about 0.6 kDa(PEO12) to about 1.2 kDa (PEO24) improved the NEP resistance ofPEGylated GANRR-CNP22(K4R) (SEQ ID NO: 36). PEGylation ofGANRR-CNP22(K4R) (SEQ ID NO: 36) to a monodispersed PEO24 moiety ratherthan a polydispersed PEG1K moiety also improved NEP resistance. Finally,although both PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) and PEG2K-CNP17have a similar total mass (keeping in mind that PEG2K is polydispersed),the former displayed substantially better NEP resistance.

NEP resistance assays were also performed on wtCNP22 and CNP variantsG-CNP37, GHKSEVAHRFK-wtCNP27 (“CNP27-HSA”, SEQ ID NO: 144) andPEO12-GANRR-CNP22(K4R) (“CNP27-PEO12”) (SEQ ID NO: 36). FIG. 19 showsthat G-CNP37 and CNP27-HSA were completely resistant to NEP cleavage,and CNP27-PEO12 exhibited much greater stability to NEP degradationcompared to wtCNP22.

Example 4 CNP Variant Stimulation of cGMP Production in NIH3T3 Cells

To determine the functional activity of CNP variants, the production ofcGMP was measured in NIH3T3 cells exposed to the CNP variants. MurineNIH3T3 cells express endogenously the CNP signaling receptor, NPR-B,which shares 98% protein sequence identity with human NPR-B. NIH3T3cells were cultured in high glucose Dulbecco's Modified Eagle Mediumsupplemented with 10% fetal calf serum and antibiotics at 37° C. with 5%CO). Twenty four to 48 hours prior to signaling, cells were passaged to12-well plates with a density of 2−5×10⁵ cells per well at the time ofthe assay. CNP variants were resuspended in 1 mM HCl to a stockconcentration of 1 mg/mL (455 uM for wtCNP22) and subsequently dilutedto a 30 uM working stock solution with phosphate-buffered saline (PBS).Ten-fold serial dilutions were prepared in phosphate-buffered saline.Culture medium was removed from the cells and replaced with 0.4 mLPBS/Dulbecco's modified Eagle medium (50/50, v/v) containing 0.75 mMisobutylmethylxanine. Plates were incubated at 37° C., 5% CO₂ for 15minutes before addition of 0.2 mL CNP variant in PBS and continuedincubation at 37° C. for 15 minutes. Reactions were stopped by theaddition of 0.2 mL lysis buffer supplied with the CatchPoint cGMP assaykit (Molecular Devices), and cGMP production was determined with theCatchPoint cGMP Assay (Molecular Devices). All stimulation experimentswere performed in duplicate.

Tables 1-4 summarize the ability of CNP variants having backbone or sidechain modifications, amino acid substitutions, N-terminal amino acidextensions, and/or N-terminal PEGylation, respectively, to stimulatecGMP production in NIH3T3 cells. In all four tables, the values for cGMPproduction in NIH3T3 cells exposed to 10 nM or 1 uM CNP variant arenormalized to cell number and cGMP production in the presence of 1 uMwtCNP22.

Regarding the results in Table 1, only Analog G having 3-Cl-Phe atposition 7 displayed substantially the same NPR-B stimulation activityat 1 uM as wtCNP22. With respect to Table 2, various CNP variants withamino acid substitutions, including Analogs AH, BO, AB, BH, BZ, BX andBR, showed substantially similar NPR-B stimulation activity as wtCNP22.

Considering the results in Table 3, many CNP variants having N-terminaland/or C-terminal modifications, including amino acid extensions,exhibited comparable NPR-B stimulation activity as wtCNP22. Thefunctional CNP variants include Analog BB, which is CNP22(G1E) attachedto heptanoic acid at the N-terminus, and Analog CD, which is the cyclicdomain of CNP22 (“CNP17” retaining the Cys6 to Cys22 sequence)conjugated to the N-terminal and C-terminal “tails” of BNP. FIG. 20illustrates that GANRR-CNP22(K4R) (SEQ ID NO: 36), CNP37 (Analog BL) andCNP53 all had similar NPR-B stimulation activity as wtCNP22 in the invitro assay.

Of note from Table 3 is that among the CNP variants assayed for both CNPfunctionality and NEP resistance, Analog AZ (R-CNP22(K4R)), Analog CC,Analog CF, Analog BL (CNP37), Analog DB (Gly-CNP37) andGHKSEVAHRFK-CNP27 (HSA-CNP27) (SEQ ID NO: 144) all had substantiallysimilar NPR-B stimulation activity as CNP22 while being substantiallymore resistant to NEP cleavage than CNP22.

With regard to the results in Table 4, nine N-terminal PEGylated CNPvariants at 1 uM stimulated cGMP production to at least about 70% of thelevel achieved by wtCNP22. Several noteworthy aspects appear in Table 4.First, N-terminal PEGylation of GANRR-CNP22(K4R) (SEQ ID NO: 36) with amonodispersed PEO polymer (PEO12 is about 0.6 kDa, PEO24 about 1.2 kDa)resulted in better NPR-B functionality than that with a polydispersedPEG polymer (PEG1K has a polymer number average molecular weight (M_(n))of around 1 kDa, PEG2K around 2 kDa) (see also FIG. 21). Second,N-terminal PEGylation of wtCNP22 with a polydispersed PEG polymer ofincreasing M_(n) (PEG1K, PEG2K, PEG5K and PEG20K) or with amonodispersed PEO polymer of greater mass (PEO12 and PEO24)correspondingly decreased the NPR-B activation ability of the CNPvariants (see also FIG. 22). Third, PEO24-GANRR-CNP22(K4R) (SEQ ID NO:36), having the N-terminal GANRR (SEQ ID NO: 8) extension, stimulatedgreater cGMP production than PEO24-CNP22 and PEO24-CNP22(K4R). Also ofnote is that among the N-terminal PEGylated CNP variants assayed forboth CNP functionality and NEP resistance, PEO12-R-CNP22(K4R),PEO12-GANRR-CNP22(K4R) (SEQ ID NO: 36) and PEO24-GANRR-CNP22(K4R) (SEQID NO: 36) all had substantially similar NPR-B stimulation activity asCNP22 while being much more resistant to NEP degradation than CNP22.

Among the CNP variants listed in Tables 1-4 and assayed for both CNPfunctionality and NEP resistance, Analogs G, BK, AZ, CC, CF, BL and DB,Pro-Gly-CNP37, HSA-CNP27 (GHKSEVAHRFK-CNP27) (SEQ ID NO: 144),PEG1K-CNP22, (PEO12)-biotin-CNP22, PEO12-R-CNP22(K4R),PEO12-GANRR-CNP22(K4R) (SEQ ID NO: 36), and PEO24-GANRR-CNP22(K4R) (SEQID NO: 36) all had substantially similar NPR-B stimulation activity aswtCNP22 while being substantially more resistant to NEP cleavage thanwtCNP22.

cGMP production assays were also carried out on wtCNP22 and CNP variantsG-CNP37, GHKSEVAHRFK-wtCNP27 (“CNP27-HSA”, SEQ ID NO: 144), wtCNP29 andPEO12-GANRR-CNP22(K4R) (“CNP27-PEO12”) (SEQ ID NO: 36). FIG. 23 showsthat CNP22 and all the CNP variants assayed induced production ofsimilar cGMP levels at either low or high dose of CNP.

Example 5 Binding Specificity for NPR-A, NPR-B and NPR-C

Signaling Competition Assay

To determine the binding specificity of CNP variants for the clearancereceptor NPR-C, a signaling competition assay is carried out. Expressionplasmids for human NPR-B or NPR-C (each purchased from OriGene) aretransiently transfected and receptors are expressed in HEK293T cells.Forty hours after transfection, NPR-B, NPR-C and native HEK293T cellsare harvested, counted and plated at a ratio of 1:1 (NPR-Bcells:competing cells (either NPR-C or native HEK293T cells)) in 12-wellor 96-well plates. Twenty hours after plating, cells are processed forthe NPR-B/cGMP stimulation assay described in Example 4. If present, thenatriuretic clearance receptor, NPR-C, is expected to bind andinternalize CNP, thereby reducing the overall CNP concentrationavailable for signaling through NPR-B, resulting in decreased cGMPproduction and a shift in the dose-response curve to the right. Arightward shift in the dose-response curve has been verified forwtCNP22. CNP variants having reduced affinity for NPR-C are not expectedto induce a shift, or are expected to induce a smaller shift, in thedose-response curve to the right. This signaling competition assay issimilar to that previously described by Cunningham (U.S. Pat. No.5,846,932; B. Cunningham, EMBO J. 13(11): 2508-2515 (1994); H. Jin etal., J. Clin. Invest. 98(4): 969-976 (1996)).

The cGMP stimulation activity of wtCNP-22, Pro-Gly-wtCNP37 and ANPthrough NPR-B and NPR-A, and their selectivity for NPR-B vs. NPR-C andfor NPR-A vs. NPR-C, were evaluated in signaling competition assays.NPR-A, NPR-B and NPR-C individually were transiently tranfected intoHEK293T cells. Thirty hours after transfection, the cells were platedinto 96-well plates: (A) 20,000 NPR-B cells+20,000 mock transfectedcells; (B) 20,000 NPR-B cells+20,000 NPR-C cells; (C) 20,000 NPR-Acells+20,000 mock transfected cells; and (D) 20,000 NPR-A cells+20,000NPR-C cells. Twenty hours after plating, the culture media was removedand replaced with serum-free media:PBS (1:1)+0.75 uM IBMX for 15minutes. For cGMP signaling through NPR-B, dose series for ANP, CNP-22and Pro-Gly-CNP37 were added and incubated at 37° C. for 12 minutesbefore the assay was stopped by cell lysis. For cGMP signaling throughNPR-A, dose series for CNP-22 and Pro-Gly-CNP37 were incubated at 37° C.for 12 minutes, while dose series for ANP were incubated at 37° C. for 6minutes (because NPR-A appeared to be a “faster” guanylyl cyclase thanNPR-B, the incubation time for ANP was shortened in order not to max out(use up all cellular GTP) too soon when signaling with ANP). FIGS. 24Aand B show that CNP-22 and Pro-Gly-CNP37 (“Pro-CNP38”) stimulated cGMPproduction through NPR-B with similar dose-response curves, and to amuch greater extent than through NPR-A, and exhibited a similar profilefor NPR-B vs. NPR-C selectivity in the signaling competition assays.

Determination of Binding Affinities (K_(i)) for NPR-A, NPR-B and NPR-C

The binding affinities (K_(i)) of CNP variants for NPR-A, NPR-B andNPR-C are determined in a heterologous competition binding assay (U.S.Pat. No. 5,846,932; B. Cunningham, EMBO J. 13(11): 2508-2515 (1994); H.Jin et al., J. Clin. Invest. 98(4): 969-976 (1996)). Membranes fromHEK293 cells, or another suitably transfectable cell line (e.g., HeLacells), expressing human NPR-A, NPR-B or NPR-C are prepared forradio-labeled ligand binding assays. Membrane preparations are dilutedin an appropriate buffer and varying concentrations of wtCNP22 or CNPvariant (competitor) are added with I¹²⁵-labeled wtCNP22 (Bachem).Samples are incubated at room temperature to allow for ligand/receptorequilibration and bound peptide is separated from free peptide byfiltration through PVDF filter membranes. Filters are washed before theaddition of scintillant and counting by a scintillation counter. Bindingis measured in duplicate for each concentration of competitor peptide.CNP variant affinity (K_(i), equilibrium dissociation constant) andB_(max), (receptor number) are calculated by non-linear regressionanalysis and/or the Cheng-Prusoff equation.

CNP variants exhibiting reduced affinity to NPR-C are expected to havereduced susceptibility to clearance by NPR-C and thus a longer plasma orserum half-life. Increased half-life of the CNP variants in circulationwould increase the availability of the variants for therapeuticactivity.

Example 6 Effect of CNP Variants on the Growth of Rat Chondrosarcoma(RCS) Cells and cGMP Production in RCS Cells

To assess the ability of CNP variants to stimulate bone growth, skeletaldysplasia is simulated in cell culture by treating rat chondrosarcoma(RCS) cells with fibroblast growth factor 2 (FGF-2), which activatesfibroblast growth factor receptor 3 (FGFR-3) and induces growth arrest(Krejci et al., J. Cell Sci., 118(21):5089-5100 (2005)).

Optimal CNP treatment parameters are determined by varying CNPconcentration (0.05, 0.1, 0.2 and 0.5 uM), and treatment duration andinterval (continuous; 2.5 min, 10 min, 30 min, 1 hr, 2 hr, 4 hr and 8 hronce a day; 2.5 min, 10 min, 30 min, 1 hr, 2 hr and 4 hr twice a day).After 72 hours, cells are counted using an automated cell counter, andthe amount of extracellular matrix is estimated using alcian bluestaining.

RCS cells are then treated with a CNP variant using the optimalconditions determined from the growth experiments with wtCNP22. Theconcentration of cGMP is measured by competitive ELISA for untreated RCScells, RCS cells treated with CNP, and RCS cells treated with the CNPvariant. Cell growth and matrix synthesis resulting from treatment withthe CNP variant are also monitored and compared to those resulting fromCNP treatment.

To assess the effect of CNP variants in a human cell culture system,primary re-differentiated human chondrocytes in alignate beads aretreated with wtCNP22 and CNP variants, and cGMP concentration isdetermined by competitive ELISA as a measure of effective CNP signaling.

The methods described herein can be employed to assess the ability ofCNP variants to stimulate cGMP production in and growth of ratchondrosarcoma cells in vitro.

Example 7 Dose Response Study in Rat Chondrosarcoma Cells

The tyrosine kinase receptor fibroblast growth factor receptor 3(FGFR-3), a negative regulator of chondrocyte growth, is contitutivelyon in achondroplasia subjects. Stimulation of the FGFR-3 receptor withFGF-2 causes growth arrest by prolonged activation of Erk MAPK, andcauses decreased matrix synthesis and loss of matrix, as well as achange in cell shape. Continuous exposure of rat chondrosarcoma (RCS)cells to fibroblast growth factor 2 (FGF-2) simulates achondroplasia incell culture by activating FGFR-3 and inducing growth arrest (Krejci etal., J. Cell Sci., 118(21): 5089-5100 (2005)). To determine the dose ofCNP variant and frequency of dosing that stimulate sufficient growth ofbone cells, a dose response study was performed using the RCS cell assayas described in Example 6.

RCS cells were seeded at 10×10³ cells per well in 24-well plates, grownfor 24 hr, treated for 72 hr, and then counted. RCS cells werecontinuously exposed to FGF-2 (5 ng/mL) to simulate a constitutivelyactive FGFR-3, which induced cell growth arrest (see bar #5 in FIG. 25).Wild-type CNP22 (0.2 uM) was cultured continuously (72 hr), 1 hr dailyor 2 hr daily. All stimulants were changed daily Continuous exposure ofRCS cells to 0.2 uM CNP22 in the presence of 5.0 ng/mL FGF-2 partiallyreversed FGF2-induced growth arrest, leading to the growth ofapproximately 200×10³ cells per well (bar #6 in FIG. 25), compared toapproximately 100×10³ cells per well in the absence of CNP22 (bar #5 inFIG. 25).

Both 1 hr exposure to CNP22 (0.2 uM) once a day and 2 hr exposure toCNP22 (0.2 uM) once a day achieved about 84% of the effect of continuousCNP22 (0.2 uM) exposure on chondrocyte growth (bars #7 and 8 in FIG.25). These results demonstrate that continuous exposure ofgrowth-arrested chondrocytes to CNP22 is not required for reversal ofcell growth arrest. Additionally, dose response studies demonstrate thatlower doses of CNP22 are capable of reversing growth arrest (FIG. 26A).

Furthermore, histological and cell morphological analysis of theextracellular matrix showed that CNP22 treatment antagonizedFGF2-mediated loss of chondrosarcoma extracellular matrix and increasedmatrix synthesis. Exposure to FGF-2 decreased matrix synthesis andincreased degradation, while addition of CNP22 to the FGF-2 cell cultureincreased matrix synthesis and partially inhibited FGF-2, as assessed by³⁵S-sulfate and ³H-Pro incorporation into, or decrease from, matrix(FIGS. 27A-D). Analysis of aggrecan and fibronectin production (mRNA andprotein) in RCS cells cultured with FGF-2 and CNP22 shows that FGF-2decreased aggrecan level and increased fibronectin level, which wasinhibited by addition of CNP22 (FIGS. 28A-C). FGF-2 induces andactivates matrix-processing molecules predominantly via Erk, andaddition of CNP22 shows some effect on this activation.

Additional high-throughput assays for measuring growth arrest, such ascrystal violet staining, are useful for measuring the effects of CNP22and variants thereof on RCS cells.

Similar dose response studies can be conducted with the CNP variantsdescribed herein to determine their effective dose for reversingFGF2-induced growth arrest of RCS cells.

Example 8A Ex Vivo Stimulation of Growth of Tibia and Femur from Micewith Mild Achondroplasia

Mouse tibial organ culture model has been used to demonstrate theefficacy of wild-type CNP22 in stimulating longitudinal bone growth.Treatment of wild-type tibiae with CNP22 at 10⁻⁸, 10⁻⁷ or 10⁻⁶ M for 6days increased longitudinal growth by 31%, 40% and 42%, respectively.Histological evaluation also showed expansion of the hypertrophic zone,e.g., an increase in the number and size of hypertrophic chondrocytes inthe growth plate (Agoston et al., BMC Dev. Biol. 7:18 (2007)). Similarfindings were observed in tibiae isolated from FGFR3^(Ach) mice (Yasodaet al., Nat. Med. 10: 80-86 (2004)).

To determine the efficacy of CNP variants in stimulating longitudinalbone growth, CNP variants were tested in a mouse organ culture model ofendochondral bone growth in wild-type mice and transgenic mice having aG380R mutation in the human FGFR-3 gene (FGFR3^(wt/Ach) heterozygote)which represent a mouse model of mild achondroplasia. In brief, thepharmacological activity of wild-type CNP22 and CNP variants wascompared in an organ culture model of embryonic or neonatal mousetibiae, isolated from wild-type and FGFR3^(wt/Ach) littermates. Overallbone growth and histological changes within the growth plate wereassessed. Conditioned culture medium is also assessed for biomarkers ofintracellular signaling (cGMP), cartilage metabolism (type II collagen,other collagens, aggrecan chondroitin sulfate), bone metabolism (bonealkaline phosphatase, osteocalcin, type I collagen [C-telopeptide,N-telopeptide]), and inflammation (interleukin-1, interleukin 6,interleukin-11).

Effective CNP variants are identified by their ability, e.g., tostimulate production of cGMP, and bone growth as measured by increasedlongitudinal bone length and expansion of the cells in the hypertrophiczone of the growth plate.

Measurement of Bone Growth

The efficacy of wtCNP22, CNP37 and PEO24-GANRR-CNP22(K4R) (SEQ ID NO:36) in stimulating longitudinal femoral growth was evaluated in themouse organ culture model. For these experiments femora were isolatedfrom 2-3 day old wild-type mice and cultured in alphaMEM supplementedwith 0.2% BSA, 0.5 mM L-glutamine, 40 units penicillin/mL and 40 ugstreptomycin/mL, for 8 days in the presence of vehicle, CNP22 or CNPvariants. The treatment commenced at day 0 and was repeated every twodays thereafter, as the medium was changed. Bones were measured prior totreatment and every two days thereafter, using a dissection microscopefitted with a 1 cm eye-piece reticule. Conditioned medium was used forbiomarker analysis. At day 8 bones were fixed in 4% paraformaldehyde for24 hr, decalcified in 5% formic acid for 24 hrs, dehydrated and embeddedin paraffin. Bones were sectioned at 5 um (microns), which were thendeparaffinized, rehydrated, and stained with Alcian Blue for 30 min (pH2.5; MasterTech). Alcian Blue stains cartilage blue. Stained sectionswere visualized and photographed by brightfield microscopy. Thethickness of the hypertrophic region of the growth plate cartilage wasdetermined by image analysis.

FIG. 29 illustrates the effect of wtCNP22, CNP37 andPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) on longitudinal growth of 3-dayold wild-type mouse femurs treated with the CNP peptides every two days.The results were normalized to measurements prior to treatment (day 0).The studies were performed in triplicate (vehicle) or quadruplicate (CNPpeptides). As shown in FIG. 29, CNP37 and PEO24-GANRR-CNP22(K4R) (SEQ IDNO: 36), as well as CNP22, were effective in stimulating longitudinalfemoral growth, with the N-terminal PEGylated CNP variant being the mosteffective.

The growth of wild-type and FGFR3^(ach) mouse femur and tibia inresponse to CNP22, CNP37 and PEO24-GANRR-CNP22(K4R) (“CNP27-PEO24”) (SEQID NO: 36) was also assessed. Culture of either wild-type orachondroplastic (FGFR3^(ach)) mouse tibia showed that CNP37 andPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) both increased the longitudinalgrowth of the tibia compared to vehicle or CNP22 (FIGS. 30 and 31).CNP22, CNP37 and PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) also stimulatedthe growth of wild-type mouse femur (FIG. 32). Moreover, each of CNP22,PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) and CNP37 increased thelongitudinal growth of FGFR3^(ach) mouse femur compared to vehicle (FIG.33).

Furthermore, ex vivo distribution of CNP37 in the growth plate ofFGFR3^(ach) mouse tibia was evaluated. Bone samples were prepared asdescribed above. Paraffin sections were cut and heat fixed for 1 hour at60° C. Antigen retrieval with 1% hyaluronidase at 37° C. (30 minutes)was followed by a 1 hour serum block (10% Normal Goat Serum). CNP22antibody (1:500 dilution; Peninsula Laboratories Inc., San Carlos,Calif.) was applied overnight at 4° C. For immunodetection, VectastainELITE ABC kit (Vector, Burlingame, Calif.) was used according to themanufacturer's recommendations. Specific bound peroxidase was visualizedby incubation with DAB substrate kit (Vector) and the reaction wasdeveloped for 3 minutes. Slides were then dehydrated and mounted andphotographed using a brightfield microscope. Staining for CNP in thegrowth plate of FGFR3^(ach) mouse tibia showed that CNP immunoreactivitywas increased in the regions of articular and hypertrophic chondrocytes(FIG. 34), indicating that CNP37 was delivered to the chondrocytes.

In addition to the distribution of CNP variants in the bone growthplate, the ex vivo effects of CNP37, CNP22 and vehicle on cells in theFGFR3^(ach) and wild-type growth plate, e.g., hypertrophic cell size andcellularity of proliferating zone, were also evaluated. Bone sampleswere prepared, including Alcian Blue staining, as described above.Images of the entire proximal growth plate were taken at 4× magnitude.The growth plate is divided into three zones, starting from theepiphyseal side of cartilage: the resting zone (individual smallchondrocytes), proliferating zone (columns of stacked chondrocytesparallel to the long axis of the bone), and hypertrophic zone (largechondrocytes and thin septa between the chondrocytes). In these regions,measurements were made by ImageJ software, including the number ofproliferating chondrocytes per column and the density of hypertrophicchondrocytes. A test square (4×4 mm²) at five different regions of thehypertrophic zone was used to determine the density of hypertrophicchondrocytes. The cell size of hypertrophic chondrocytes was calculatedby 1 over determined cell density. Cellularity of proliferating columnswas increased by CNP37 and CNP22 in both wild-type and FGFR3^(ach) mice(FIGS. 35B and C). Chondrocyte hypertrophy in FGFR3^(ach) mice was alsoincreased as a result of culture with CNP22 or CNP37 (FIGS. 36B and C).

Ex vivo studies of cultures of mouse bones indicated that CNP37 wasdelivered to the growth plate and was able to increase chondrocytecellularity and hypertrophy, which are associated with growth plateexpansion and longitudinal bone growth. To assess the biodistribution ofCNP37 in the bone growth plate in vivo and the in vivo effects of CNP37on the growth plate (including total growth plate thickness,hypertrophic zone thickness, and cellularity of proliferating zone),bone samples were obtained from FGFR3^(ach) mice treated with vehicle orCNP37 as described above. For biodistribution and in vivo effectsstudies, tibias were fixed and stored in 70% ethanol. Forimmunohistochemistry, the samples were decalcified in 5% formic acid for2 days, dehydrated and embedded in paraffin. Bones were sectioned at 5um (microns), which were then deparaffinized, rehydrated, and used forCNP immunohistochemistry as described above. For cellular imageanalysis, bones were sectioned at 5 (microns), and then deparaffinized,rehydrated, and stained with Alcian Blue for 30 minutes (pH 2.5;MasterTech) and Hematoxylin & Eosin for 30 seconds. Stained sectionswere visualized and photographed by brightfield microscopy. Thicknessesof the growth plate and the proliferating and hypertrophic zones weremeasured using ImageJ software.

In vivo biodistribution studies demonstrated that, similar to the exvivo studies, CNP immunoreactivity was increased in the regions ofarticular and hypertrophic chondrocytes in the tibia growth plate ofFGFR3^(ach) mice treated with CNP37, indicating that CNP37 was deliveredin vivo to the growth plate of FGFR3^(ach) mouse tibia (FIG. 37).Furthermore, CNP37 treatment significantly increased the total growthplate thickness, proliferating zone thickness and hypertrophic zonethickness of FGFR3^(ach) mouse tibia in vivo (FIGS. 38A-C).

These results demonstrate that CNP variants of the disclosure penetrateinto the growth plate of wild-type and achondroplastic animals, increasethe number and size of chondrocytes, increase the thickness of theproliferating zone and the hypertrophic zone of the growth plate, andincrease longitudinal bone growth in treated wild-type andachondroplasic animals. Therefore, the CNP variants are useful forstimulating bone growth in achondroplastic subjects.

Measurement of Biomarkers

In addition to measurement of bone growth in response to CNP variants,assay of the levels of biomarkers for cartilage and bone formation andgrowth induced in response to CNP variants is useful for evaluating theeffect of CNP variants on bone growth.

Femurs and tibias were isolated from wild-type and FGFR3^(ach) mice asdescribed above. Bones were cultured with CNP22 or a variant thereof foreight days with the replacement of media every two days. On the eighthday, media was collected and analyzed for the biomarkers cGMP (cyclicguanosine 3′, 5′ cyclic monophosphate) and fragments of cleaved collagentype II, a cartilage-specific marker for cartilage turnover. Bothmarkers were measured using commercially available enzyme-linkedimmunosorbent assays (ELISA) for cGMP (Cayman Chemical Co., Ann Arbor,Mich.) and cleaved collagen type II (Cartilaps) (ImmunodiagnosticSystems, Fountain Hills, Ariz.), following the manufacturer's protocol.

Levels of cGMP and collagen type II fragments were measured from cellculture extracts after exposure to CNP22, CNP37 orPEO24-GANRR-CNP22(K4R) (“CNP27-PEO24”) (SEQ ID NO: 36). FIGS. 39-42 showa large increase (p<0.01) in the levels of cGMP in the media afterexposure of ex-planted wild-type and FGFR3^(ach) mouse femurs and tibiasto CNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36). In addition,exposure of wild-type and FGFR3^(ach) mouse femurs to CNP22, CNP37 orPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) increased the levels of cleavedcollagen type II, with the treated FGFR3^(ach) mouse femurs showingsignificantly increased (p<0.05) levels of collagen type II fragments(FIG. 43). Elevated levels of collagen type II fragments indicate aturnover of the cartilage matrix, and cartilage turnover typicallyprecedes new bone formation in growing bones.

Example 8B Ex Vivo Stimulation of Growth of Femur from Mice with SevereAchondroplasia

The effect of a CNP variant on the growth of bones from mice with severeachondroplasia was evaluated ex vivo. Used in the study were transgenicmice expressing a human FGFR-3gene having a Y367C mutation(FGFR3^(Y367C)) [S. Pannier et al., Biochim. Biophys. Acta, 1792(2):140-147 (2009)], which represent a mouse model of severe achondroplasia.Femurs were isolated at embryonic day 16.5 and cultured for 6 days inthe presence of 1 uM Pro-Gly-CNP37. Bone lengths were measured at Day 1and Day 7. The bones were then paraffin-embedded, sectioned and stainedwith hematoxylin and eosin to assess histological changes and cellularmorphology. Treatment of bone explants isolated from FGFR3^(Y367C) micewith Pro-Gly-CNP37 (“ProCNP38”) resulted in increase in bone growth andexpansion in the growth plate (FIG. 44). Femurs from FGFR3^(Y367C) micetreated with vehicle for 6 days showed an 18% deficiency in growthlength-wise compared to wild-type femurs treated with vehicle. Treatmentof femurs from FGFR3^(Y367c) mice with 1 uM Pro-Gly-CNP37 for 6 daysreduced the growth deficiency to just 11%, i.e., reduced the growthdefect by around 40%.

Example 9 Serum/Plasma Stability of CNP Variants In Vitro

In preparation for pharmacokinetics (PK) studies, the stability of CNPvariants in serum and/or plasma is evaluated.

Briefly, the analyte is isolated by the removal of serum or plasmaproteins by either a 2% trichloroacetic acid precipitation or a 1:3serum:acetonitrile precipitation. The precipitation mixture is vortexedat 14,000 rpm for five minutes, and a portion of the supernatant isremoved and diluted with water prior to transfer to a silanizedautosampler vial for analysis. Serum extracts are then analyzed byreverse-phase high performance liquid chromatography (RP-HPLC) withelectrospray ionization mass spectrometry (ESI-MS). A single mass (m/z),shown to be specific for the CNP variant, is monitored for quantitationpurposes.

Initially, analytical stability and recovery is determined. Analytical(RP-HPLC and ESI-MS) parameters are optimized through the analysis ofmatrix standards (serum extracts fortified with analytepost-precipitation). After optimization, analytical recovery isdetermined by spiking serum samples at known concentrations andcomparison of the analyte response to that of matrix standards preparedat similar concentrations. Analyte stability in serum extracts is alsodetermined to assure no significant losses occur after serumprecipitation and prior to actual analysis. To test the effect offreezing on serum stability, a two-cycle freeze/thaw study is alsoperformed. In this study a serum sample is spiked with CNP variant andanalyzed prior to freezing overnight at −20° C. The sample is thenthawed at room temperature and re-analyzed. The process is repeated fora second freeze/thaw cycle.

Serum stability of CNP variant is determined by spiking of serum/plasmasamples with CNP variant at a concentration of 10 ug/mL. The sample isplaced in a 37° C. water bath for a period of three hours. At 30 minuteintervals duplicate aliquots of serum are removed and analyzed. If rapidlosses of analyte are evident (>50% in 30 minutes), the study may berepeated with 10 minute timepoints.

In an exemplary method for determining the stability of CNP variants inmurine plasma, a mixture of CNP variant (10 uL of a stock solution ofabout 2.5-5.0 mg/mL), heparinized murine plasma (50 uL, Bioreclamation,CD-1 Lith Hep 62231), and 5 M NaCl (10 uL) is incubated at 37° C. and 5%CO₂ for 0-5 hr, and then quenched with 10× protease inhibitor cocktail(15 uL, Sigma P2714). For extraction, 150 uL of MeOH/0.1% FA is added to85 uL of the reaction mixture, and the resulting mixture is vortexed for1 min and then centrifuged at 15° C. for 15 min. 75 uL of thesupernatant is added to 300 uL of aqueous 0.1% FA. A small portion ofthe resulting mixture is subjected to analysis by LC/MS.

Example 10 Pharmacokinetics and cGMP Production in Rats and Mice

Studies were conducted in normal rats to evaluate the pharmacokinetics(PK) profile of CNP22 and certain CNP variants and the time courses ofplasma cGMP concentration after single intravenous (i.v.) orsubcutaneous (s.c.) administration of the CNP peptides. Plasma CNPimmunoreactivity was determined by using a competitive radioimmunoassay(RIA) with an anti-CNP rabbit polyclonal antibody. Plasma cGMPconcentration was determined by RIA using a commercially available kit(YAMASA cyclic GMP Assay kit, YAMASA Corporation).

Normal male rats, 7-8 weeks of age, were used. Recombinant wild-typeCNP22, CNP37 and PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) were evaluated.A dosage of 20 nmol/kg of each CNP peptide as a solution in 5% mannitolwas intravenously injected once into the tail, or a dosage of 50 nmol/kgof each CNP peptide as a solution in 0.03 mol/L acetic acid buffersolution, pH 4.0, containing 1% (w/v) benzyl alcohol and 10% (w/v)sucrose, was subcutaneously injected once into the back.

Plasma CNP immunoreactivity was determined by the competitive RIA usinganti-CNP rabbit polyclonal antibody. Standard and QC samples wereprepared. Fifty uL of the standard, QC and assay samples were added,respectively, to test tubes containing 50 uL of RIA buffer. Dilutedanti-CNP rabbit polyclonal antibody (100 uL) was added to the tubes. Alltubes were kept at 4° C. overnight. ¹²⁵I-[Tyr⁰]-CNP22 solution (100 uL)and rabbit IgG solution (100 uL) were added and left at approximately 4°C. overnight. One milliliter of anti-rabbit IgG goat serum containing10% polyethylene glycol was added, vortexed and left at approximately 4°C. for at least 1 hour, and then the insoluble fraction was precipitatedby centrifugation. After aspiration of the supernatant, the amount ofradiation (gamma line) in the sediment was measured by a gamma-counter.Each sample was measured in duplicate, and the mean was adopted as thevalue determined.

Plasma cGMP concentrations in the sample at 5, 30, 60 and 90 minutesafter i.v. dosing, or at 5, 30, 60, 120 and 180 minutes after s.c.dosing, were determined by the competitive RIA using anti-cGMPmonoclonal antibody. Standard samples were prepared. 100 uL of the assaysamples (standard solutions for the calibration curve or the dilutedplasma samples for cGMP determination) were transferred to test tubes.Then 100 uL of anti-cGMP monoclonal antibody solution and 100 uL of¹²⁵I-labeled succinyl cGMP tyrosine methyl ester solution were added tothe tubes, respectively. All tubes were kept at 4° C. overnight. Afterthe addition of 500 uL dextran charcoal solution, the tubes werevortexed and then placed on ice for 10 minutes. The reaction mixture wascentrifuged and 500 uL of the supernatant was transferred from eachsample to a new test tube. The amount of radiation (gamma line) in thesupernatant was measured by a gamma-counter. Each sample was measured induplicate, and the mean was adopted as the value determined.

Plasma CNP immunoreactivity was employed for pharmacokinetics (PK)analysis. PK analysis was performed using WINNONLIN® Professional(Pharsight Corporation). The PK profiles of CNP22, CNP37 andPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) after i.v. administration werecalculated using PK parameters such as concentration at 0 hour (C₀:extrapolation, pmol/mL), total body clearance (CL_(tot): mL/min/kg),distribution volume at steady state (V_(dss): mL/kg), area under theplasma concentration-time curve (AUC: pmol·min/mL), mean residence time(MRT: min), and half-life (T_(1/2): min). The PK profiles of the CNPpeptides after s.c. administration were calculated using PK parameterssuch as maximum plasma concentration (C_(max): pmol/mL), time to reachC_(max) (T_(max): min), area under the plasma concentration-time curve(AUC: pmol·min/mL), mean residence time (MRT: min), and half-life(T_(1/2): min).

In plasma spike recovery experiments, the RIA detected CNP22, CNP37 andPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) similarly (data not shown).

Procedures similar to those described above are employed to study inmice the PK profiles of CNP22 and variants thereof and their ability tostimulate cGMP production.

The PK profiles of CNP22, CNP37 and PEO24-GANRR-CNP22(K4R) (SEQ ID NO:36) after i.v. administration in three rats are illustrated in FIG. 45.As shown by FIG. 45, CNP37 and PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36)had a much longer half-life and a much greater bioavailability thanCNP22. The half-life, T_(1/2) (min), was 1.42 (±0.45) for CNP22, 22.3(±1.5) for PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36), and 49.5 (±28.0) forCNP37. The area under the curve, AUC (pmol·min/mL), was 320 (±54) forCNP22, 1559 (±568) for CNP37, and 2084 (±424) for PEO24-GANRR-CNP22(K4R)(SEQ ID NO: 36).

The PK profiles of the three CNP peptides after s.c. administration inthree rats are depicted in FIG. 46. Compared to CNP22,PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) had a much longer half-life (78.1min (±16.4) vs. 10.0 (±5.0)) and a much greater bioavailability (60%(±6%) vs. 19% (±9%)).

The time courses of plasma cGMP concentrations after i.v. administrationof the three CNP peptides in three rats are displayed in FIG. 47. FIG.47 clearly demonstrates that i.v. administration of CNP37 andPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) resulted in much higher plasmalevels of cGMP at 30, 60 and 90 minutes than i.v. administration ofCNP22.

The time profiles of plasma cGMP concentrations after s.c.administration of the three CNP peptides in three rats are shown in FIG.48. Subcutaneous administration of PEO24-GANRR-CNP22(K4R) (SEQ ID NO:36) and CNP37 also resulted in substantially higher plasmaconcentrations of cGMP than s.c. administration of CNP22, with thedifference relative to CNP22 increasing over time forPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36), but decreasing over time forCNP37.

The rat studies indicate that compared to wtCNP22, the CNP variantsCNP37 and PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) had a substantiallylonger half-life in vivo, had a substantially greater bioavailability invivo, and stimulated substantially higher levels of cGMP production invivo for an extended period of time. The resistance of CNP37 andPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) to NEP degradation correlates toa longer plasma half-life in vivo, which in turn correlates to prolongedNPR-B/cGMP signaling in vivo. These results show that compared to CNP22,the CNP variants of the disclosure, administered by i.v. or s.c.injection (e.g., once daily), can be more effective in treatingCNP-responsive conditions or disorders, such as bone-related disordersand vascular smooth muscle disorders.

Example 11 Pharmacokinetics Study in Mice

To determine CNP variants having increased NEP resistance, for efficacystudy in FGFR3^(ach) mice (see Example 13), a pharmacokinetics (PK)study is carried out that compares the pharmacokinetics properties ofCNP variants to wild-type CNP22. The FGFR3^(ach) mouse is a mutant mousemodel of mild achondroplasia, containing a single transgene on abackground of FVB mice.

Wild-type CNP22 or variant thereof is administered as a singleintravenous (i.v.) dose in 6-week old wild-type FVB mice. Exemplary PKstudies were conducted using wtCNP22. Six-week old FVB/N mice wereintravenously administered wtCNP22 in a single dose at 100 nmol/kg. Meanplasma levels of CNP22 were calculated, and the estimated half-life ofCNP22 was determined to be from 0.76 min to 1.03 min.

CNP variants displaying greater resistance to NEP degradation areexpected to exhibit increased serum concentrations over time and alonger half-life in vivo.

Example 12 Efficacy of CNP Variants in Wild-Type Mice

The in vivo effects of CNP variants on bone growth were assessed inwild-type mice. Three week old FVB wild-type male mice received dailysubcutaneous (s.c.) injection of either vehicle, G-CNP37 (200 nmol/kg)or PEO12-GANRR-CNP22(K4R) (“CNP27-PEO12”) (SEQ ID NO: 36) (200 nmol/kg)for 5 weeks. Body weight was measured at least once weekly. Tail lengthwas measured at least once weekly using digital caliper readings, andbody length (naso-anal length), bone length (tibia, femur, ulna andhumerus), skull length (anterior to posterior cranial segment) andlumbar vertebrae 5 (LV5) length were measured after 5 weeks of treatmentusing a caliper. X-rays were taken at baseline and after 5 weeks oftreatment.

Five weeks of treatment of wild-type mice with G-CNP37 resulted insignificant body weight gain, with increased body weight being observedbeginning at Day 9 (p<0.05) (FIG. 49). Treatment with G-CNP37 alsoresulted in significantly increased tail length, beginning at the secondweek after treatment (p<0.01) (FIG. 50).

Table 10 shows the percentage change in tail length, body (naso-anal)length, skull (anterior to posterior cranial segment) length, bone(femur, tibia, humerus and ulna) lengths, and lumbar vertebrae 5 (LV5)length in wild-type mice injected s.c. once daily with 200 nmol/kg ofeither G-CNP37 or PEO12-GANRR-CNP22(K4R) (“CNP27-PEO12”) (SEQ ID NO: 36)for 5 weeks, relative to a value of 100% for wild-type mice treated withvehicle only.

TABLE 10 Anterior to Naso- Posterior Tail Anal (Skull) Femur TibiaHumerus Ulna LV5 G-CNP37 119%** 114%** 104%* 109%** 105%** 104%** 101%108%* CNP27- 101% 104%** 101% 102%* 100% 102%*  96%* 103% PEO12 **p <0.01, *p < 0.05Treatment with G-CNP37 resulted in significantly increased tail length,body (naso-anal) length, skull length, proximal bone (femur and humerus)length, distal bone (tibia) length, and vertebral (lumbar vertebrae 5)length compared to treatment with vehicle.

Lower doses of Pro-Gly-CNP37 dosed daily s.c. at 5 nmol/kg, 20 nmol/kgor 70 nmol/kg for five weeks resulted in a dose-dependent increase intail length, body (naso-anal) length, and bone lengths compared tovehicle. Table 11 shows the percentage change in tail length, body(naso-anal) length, and bone (femur, tibia, humerus and ulna) lengths inwild-type mice injected s.c. once daily with 5 nmol/kg, 20 nmol/kg or 70nmol/kg Pro-Gly-CNP37 for 5 weeks, relative to a value of 100% forwild-type mice treated with vehicle only.

TABLE 11 Naso- Tail Anal Femur Tibia Humerus Ulna  5 nmol/kg 107.6%**102.7%** 103.3%** 101.7%** 101.1% 100.9%* 20 nmol/kg 107.8%** 107.6%**107.1%** 103.4%** 102.3%** 102.5%** 70 nmol/kg 113.6%** 112.5%**109.3%** 105.9%** 103.6%** 104.1%** **p < 0.01, *p < 0.05

In another study, various dosing regimens of Pro-Gly-CNP37 wereadministered for nine weeks followed by one week of recovery. Wild-typeFVB mice were dosed s.c. with:

-   (1) vehicle daily for nine weeks, followed by one week of recovery;-   (2) 20 nmol/kg Pro-Gly-CNP37 daily for one week, followed by three    doses per week for eight weeks and one week of recovery;-   (3) 20 nmol/kg Pro-Gly-CNP37 on alternating weeks; or-   (4) 5 nmol/kg Pro-Gly-CNP37 daily for nine weeks followed by one    week of recovery.

Increased growth in tail length, body length, and bone lengths wasobserved in all treatment groups (Groups 2, 3 and 4) at the end of thestudy. Table 12 shows the percentage change in tail length, body(naso-anal) length, and bone (femur, tibia, humerus and ulna) lengths inwild-type mice administered Pro-Gly-CNP37 (“Pro-CNP38”) under the doseregimens described above, relative to a value of 100% for wt micetreated with vehicle only.

TABLE 12 Total Pro-CNP38 received Naso- (nmol/kg) Tail Anal Femur TibiaHumerus Ulna Group 2 620 104.8%** 106.0%** 105.3%** 100.8% 102.5% 101.7%Group 3 700 106.7%** 106.5%** 104.1%** 102.5%** 102.6%* 102.8%* Group 4350 104.9%** 105.1%** 107.3%** 102.7%** 102.3%** 103.6%** **p < 0.01, *p< 0.05While all dose regimens of Pro-Gly-CNP37 increased axial andappendicular growth parameters measured, daily dosing of Pro-Gly-CNP37promoted appendicular growth (femur, tibia, humerus, and ulna) at alower total dose level (Group 4) compared to regimens with less frequentdosing (Groups 2 and 3).

Example 13 Efficacy in Mouse Model of Mild Achondroplasia

The efficacy of CNP variants in enhancing growth and correctingachondroplasia was tested in a mouse model of mild achondroplasia, usinga strain of transgenic mice expressing a human FGFR-3 gene having aG380R mutation (FGFR3^(ach)) (Wang et al., Proc. Natl. Acad. Sci. USA,96(8): 4455-4460 (1999); Naski et al., Development USA, 125: 4977-4988(1998); U.S. Pat. Nos. 6,265,632 and 6,136,040).

At 3 weeks of age, FGFR3^(ach) mice and their wild-type littermates wereanesthetized to have lateral whole-body X-ray images taken by Faxitron,and randomized by body weight into the following treatment groups(n=8/group): (1) wild-type/vehicle, (2) FGFR3^(ach)/vehicle, (3)FGFR3^(ach)/CNP37, and (4) FGFR3^(ach)/PEO24-GANRR-CNP22(K4R) (SEQ IDNO: 36). The mice received once daily subcutaneous (s.c.) administrationof designated test article (vehicle or 200 nmol/kg CNP variant) for 5weeks. Satellite groups (n=3) were used to confirm exposure of each testarticle following a single subcutaneous administration on Day 1.Wild-type male FVB mice that received daily s.c. injection of vehiclefor 5 weeks were used as a control for normal growth.

X-ray measurements at baseline and at the end of the study wereperformed to determine change in the head length, area of the skull, andthe external auditory meatus (EAM, the ear canal running from the outerear to the middle ear). Body weight and tail length were measured atleast once weekly, using a digital caliper to measure tail length, andbody (naso-anal) length was measured after 5 weeks of treatment. Bone(tibia, femur, ulna and humerus) lengths were measured using a digitalcaliper at necropsy.

On Day 37, all mice were sacrificed by terminal anesthesia andwhole-animal photographs and X-ray images by Faxitron were taken. Leftand right tibia, femur, humerus, and ulna were collected and measuredusing a digital caliper. The left portions of each bone were processedfor histology, and the right portions were snap-frozen for archival.Samples obtained from the bones were used to evaluate the effects of CNPvariants on endochondral bone growth.

Treatment of FGFR3^(ach) mice with CNP37 by once daily s.c. injectionfor 5 weeks resulted in significantly increased body length (FIG. 51),tail length (FIG. 52), distal bone (ulna and tibia) lengths (FIGS. 53Aand B), and proximal bone (humerus and femur) lengths (FIGS. 54A and B).Moreover, treatment with CNP37 increased the head length (FIG. 56), areaof the external auditory meatus (FIG. 57), and spine length (FIG. 58)via extension of the vertebral bodies. Additionally, treatment withCNP37 corrected rhizomelia (disproportion of the length of the proximallimbs) of FGFR3^(ach) mice, i.e., restored proportional growth ofproximal bones, as assessed by the femur:tibia ratio (FIG. 55). Table 13summarizes the percentage change in tail length, body (naso-anal)length, and bone (femur, tibia, humerus and ulna) lengths in FGFR3^(ach)mice injected s.c. once daily with 200 nmol/kg of either CNP37 orPEO24-GANRR-CNP22(K4R) (“CNP27-PEO24”) (SEQ ID NO: 36) for 5 weeks,relative to a value of 100% for FGFR3^(ach) mice treated with vehicleonly.

TABLE 13 Naso- Tail Anal Femur Tibia Humerus Ulna CNP37 115%** 109%**112%** 107%** 105%* 106%** CNP27- 100%  99% 100% 102% 100% 101% PEO24**p < 0.01, *p < 0.05

The results of these studies show that CNP37 can stimulate spinal andlong bone growth, help to correct rhizomelia by preferentiallyincreasing the length of the femur over the tibia, and help to restorecraniofacial proportions in FGFR3^(ach) mice. These results indicatethat CNP37 and potentially other CNP variants may be effective incorrecting the symptoms of achondroplasia and treating subjects havingdefects in bone growth or in need of increased bone growth.

Example 14 Measurement of Biomarkers and Evaluation of Immunogenicity inWild-Type and Achondroplastic Mice

Measurement of Biomarkers After CNP Administration

The levels of bone growth biomarkers were measured in wild-type andachondroplastic (FGFR3^(ach)) mice.

Transgenic FVB FGFR3^(ach) mice (a mouse model of mild achondroplasia)were treated daily for 5 weeks by subcutaneous injection with eithervehicle (30 mM acetic acid/acetate buffer, 1% benzyl alcohol, 10%sucrose, pH 4.0), CNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (“CNP27-PEO24”)(SEQ ID NO: 36) at 200 nmol/kg for each CNP compound, as describedabove. Plasma and serum were collected during the study. Harvestedplasma was stored with 10× protease inhibitor at −80° C. until analysis.cGMP levels were measured 15 minutes post-injection on Day 36 fromK2-ETDA plasma collection tubes. Fragments of cleaved collagen type II(cartilage-associated biomarkers), osteocalcin (bone-associatedbiomarker), and IgG (relating to immunogenicity) were measured fromterminal bleed serum at the end of the study (Day 37). cGMP was measuredusing a commercially available ELISA kit (Cayman Chemical Co., Cat. No.581021.1), cleaved collagen type II (Cartilaps) was measured using acommercially available kit from Immunodiagnostic Systems (Cat. No.3CAL4000), and osteocalcin was measured using a commercially availablekit from Biomedical Technologies Inc. (Stoughton, Mass.).

FIG. 59 shows an increase in cGMP plasma levels in FGFR3^(ach) micetreated with CNP37 or PEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) compared tovehicle. FIGS. 60 and 61 show that administration of CNP37 resulted inthe greatest elevation of serum levels of cleaved collagen type II andosteocalcin. These results indicate that administration of the CNPpeptides, in particular CNP37, to FGFR3^(ach) mice led to increasedlevels of bone growth markers, suggesting increased bone formation andgrowth in CNP peptide-treated FGFR3^(ach) mice.

For evaluation of biomarkers in wild-type mice, wild-type FVB mice weretreated daily for 5 weeks by subcutaneous injection with either vehicle(30 mM acetic acid/acetate buffer, 1% benzyl alcohol, 10% sucrose, pH4.0), 200 nmol/kg G-CNP37, 200 nmol/kg PEO12-GANRR-CNP22(K4R)(“CNP27-PEO12”) (SEQ ID NO: 36), or 20 nmol/kg or 70 nmol/kgPro-Gly-CNP37. Plasma and serum were collected during the study.Harvested plasma was stored with 10× protease inhibitor at −80° C. untilanalysis. cGMP levels were measured 15 minutes post-injection on Day 36from K2-ETDA plasma collection tubes. The levels of cleaved collagentype II, bone-specific alkaline phosphatase, and IgG (relating toimmunogenicity) were measured from terminal bleed serum at the end ofthe study (Day 37). cGMP and cleaved collagen type II (Cartilaps) weremeasured as described above. Bone-specific alkaline phosphatase wasmeasured using a commercially available kit (Cusabio, Cat. No.CSB-E11914m).

Administration of G-CNP37 significantly increased (p<0.05) the level ofcGMP (FIG. 62) and particularly the level of fragments of cleavedcollagen type II (FIG. 63) in wild-type mice compared to vehicle. Thesignificantly higher level of collagen type II fragments resulting fromadministration of G-CNP37 indicates turnover of the cartilage matrix,suggesting that G-CNP37 stimulated new bone formation in growing bonesin wild-type mice.

Both doses of Pro-Gly-CNP37 (“Pro-CNP38”), 20 and 70 nmol/kg,significantly increased (p<0.05) plasma cGMP level 15 minutes afteradministration compared to wild-type mice treated with vehicle (FIG.64). Administration of the higher dose (70 nmol/kg) of Pro-Gly-CNP37also significantly increased (p<0.05) the level of cleaved collagen typeII compared to vehicle-treated mice (FIG. 65), suggesting that thehigher dose of Pro-Gly-CNP37 stimulated cartilage matrix turnover priorto new bone formation in wild-type mice. Furthermore, administration ofthe higher dose (70 nmol/kg) of Pro-Gly-CNP37 increased (p<0.05) thelevel of bone-specific alkaline phosphatase compared to vehicle-treatedmice (FIG. 66), suggesting that the higher dose of Pro-Gly-CNP37increased bone remodeling in wild-type mice.

Evaluation of Immunogenicity of CNP Variants

Because the CNP variants are peptide derivatives, it is possible thatadministration of the peptides may lead to an immunogenic response invivo. To assess whether an immune response occurred after successiveadministrations of CNP variant, measurement of serum antibody levels wasperformed.

An IgG assay was performed to assess whether an IgG immune response wastriggered by 5 week exposure of achondroplastic FGFR3^(ach) mice toCNP22, CNP37 or PEO24-GANRR-CNP22(K4R) (“CNP27-PEO24”) (SEQ ID NO: 36).IgG is the most predominant immunoglobulin in mouse and human serum andis produced as part of the secondary immune response to an antigen. TheIgG response to administration of the CNP peptides was determined asfollows. 96-well plates were coated with 100 ng/mL CNP22, CNP37 orPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) in BupH PBS buffer(Pierce/Thermo, Cat. No. 28372, Rockford, Ill.). After overnightincubation, plates were blocked with Casein-PBS blocking buffer(Pierce/Thermo, Cat. No. 37528) for two hours at room temperature withshaking at 300 rpm. After washing with wash buffer (1×PBS with 0.05%Tween), diluted serum samples from a terminal bleed (diluted 1:25) wereadded to the plate. Positive and negative controls were also loaded ontothe plate. Positive control was 1:25 diluted serum (pooled from 6individual FVB mice) with anti-CNP22 antibody added (Bachem rabbitanti-CNP22 IgG, Cat. No. T-4222) at 1:1000 dilution. Negative controlwas the diluted pooled serum. After two hours of incubation, the plateswere washed and secondary antibody diluted in blocking buffer was addedto the wells. For the mouse serum samples, anti-mouse IgG Fcγ(peroxidase-conjugated affini-pure goat anti-mouse IgG, Fcγ fragment,Cat. No. 115-035-071, Jackson Immunoresearch, West Grove, Pa.) was addedat a 1:10,000 dilution. For the positive and negative controls,anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Cat. No. SC-2004, SantaCruz, Calif.) was added to blocking buffer. After two hours ofincubation at room temperature with shaking at 300 rpm, plates werewashed with wash buffer. 100 uL of TMB (One-step TMB, Pierce/Thermo,Cat. No. 34022) was added to all wells. Plates were incubated at roomtemperature for 15 minutes with shaking at 300 rpm. Colorimetricreactions were stopped by the addition of 100 uL of 2 N H₂SO₄. Plateswere read at 450 nm (Spectramax, Molecular Devices, Sunnyvale, Calif.)and data was analyzed using SoftMax Pro software (Molecular Devices).

Serum samples from FGFR3^(ach) mice treated with CNP22 orPEO24-GANRR-CNP22(K4R) (SEQ ID NO: 36) indicated no positive IgG immuneresponse in any of the mice. Only one out of nine CNP37-treatedFGFR3^(ach) mice showed a slightly positive IgG response.

The immunogenic response of wild-type mice administered G-CNP37,PEO12-GANRR-CNP22(K4R) (SEQ ID NO: 36) or Pro-Gly-CNP37, as measured byan increase in serum IgG level, was also assessed by examining terminalbleed serum samples as described above. None of the wild-type micetreated with PEO12-GANRR-CNP22(K4R) (SEQ ID NO: 36) or Pro-Gly-CNP37 (20or 70 nmol/kg) showed a positive IgG immune response, and only one outof six wild-type mice administered G-CNP37 showed a slightly positiveIgG response.

Measurement of Biomarkers Under Different CNP Dosing Regimens

Wild-type FVB mice were treated daily for 9 weeks by subcutaneous (s.c.)injection of vehicle (30 mM acetic acid/acetate buffer, 1% benzylalcohol, 10% sucrose, pH 4.0) or Pro-Gly-CNP37 (“Pro-CNP38”) underdifferent dosing regimens. Group 1 included vehicle-treated miceinjected s.c. daily for 9 weeks followed by 1 week of no treatment.Group 2 included mice treated with Pro-Gly-CNP37 (20 nmol/kg) once dailyfor 1 week, followed by three doses per week for 8 weeks, followed by 1week of no treatment. Group 3 contained mice treated with Pro-Gly-CNP37(20 nmol/kg) once daily in alternate weeks (weeks 1, 3, 5, 7 and 9),with 1 week of no treatment following each week of treatment. Group 4contained mice treated with Pro-Gly-CNP37 (5 nmol/kg) once daily for 9weeks followed by 1 week of no treatment. Finally, Group 5 included micetreated with Pro-Gly-CNP37 (5 nmol/kg) once daily for 5 weeks withoutany week of no treatment. Terminal bleed serum was collected from themice, and cleaved collagen type II, total alkaline phosphatase and totalantibody levels were measured therefrom. Cleaved collagen type II levelswere measured as described above. Total alkaline phosphatase levels,predominantly from the liver and bones, were measured with theassistance of a veterinary diagnostic laboratory testing facility(Antech).

A total antibody assay was developed to evaluate potential immuneresponse. The platform used for the total antibody assay was anelectrochemiluminescent assay (ECLA). The ECLA platform utilizes abiotin-labeled drug (here, biotin-Pro-Gly-CNP37) and a ruthenium-labeleddrug (here, Ru—Pro-Gly-CNP37). The biotin-labeled drug binds to astreptavidin-coated plate that contains electrodes, and theruthenium-labeled drug functions as the detection component of theassay, as ruthenium can be electrochemically stimulated. A drug-specificantibody (here, a CNP-specific antibody) binds to the biotin-labeleddrug and the ruthenium-labeled drug and “bridges” the two labeled drugs.Among the advantages of the ECLA platform, any isotype of antibody (IgG,IgM, etc.) can be detected, and the ECLA assay is species-independent.

The total antibody assay was performed as follows. Pro-Gly-CNP37 waslabeled with biotin in a 4:1 challenge ratio, and Pro-Gly-CNP37 was alsolabeled separately with ruthenium in a 10:1 challenge ratio. Bothseparate labeling reactions were quenched by the addition of glycine,and the samples from both reactions were buffer-exchanged to PBS. Lowand high QCs were prepared by using commercially available anti-CNP22antibody (Bachem rabbit anti-CNP22 IgG, Cat. No. T-4222) added at lowand high concentrations to 5% diluted FVB mouse serum. Mouse serumsamples were diluted to 5% using assay diluent (5% BSA in PBS, MSD Cat.No. R93AA-1). A working solution was prepared by adding biotin-labeledPro-Gly-CNP37 to assay diluent, then adding ruthenium-labeledPro-Gly-CNP37 to assay diluent, and then combining the two solutionstogether. Twenty-five uL of low and high QCs were loaded onto the plateas controls. Then 25 uL of sample was added to a non-binding 96-wellplate, followed by the addition of 50 uL of working solution to allwells. Samples and QCs were incubated with the working solution at roomtemperature for 2 hours with shaking at 350 rpm. In the meantime, an MSDstreptavidin plate (MSD Cat. No. L13SA-1) was blocked with blockingbuffer (MSD Cat. No. R93AA-1) at room temperature for 2 hours withshaking at 350 rpm. At the end of the two hour incubation, the MSDstreptavidin plate was washed, and then 50 uL of sample or QC wastransferred to the MSD plate. The plate was then incubated at roomtemperature for 1 hour with shaking at 350 rpm. At the end of the 1 hourincubation, the MSD plate was washed and 150 uL of 2× read buffer (MSDCat. No. R92TC-2) was added to the plate. The plate was read using anMSD PR400 machine.

All five groups of mice exhibited substantially comparable levels ofcleaved collagen type II (FIG. 67). Treatment of the mice in Group 5with 5 nmol/kg Pro-Gly-CNP37 once daily for 5 weeks significantlyincreased (p<0.001) total alkaline phosphatase level (FIG. 68), which isindicative of bone remodeling. Serum samples from each of the fourgroups of mice treated with Pro-Gly-CNP37 did not show a positiveantibody response in the total antibody assay.

Not to be bound by any theory, there are possible explanations as to whythe four groups of CNP-treated mice did not exhibit statisticallysignificant difference in cleaved collagen type II level compared tovehicle-treated mice, and why only the CNP-treated mice in Group 5exhibited a statistically significant increase in total alkalinephosphatase level compared to vehicle-treated mice. It is possible thatbecause the vehicle-treated mice in the study were also growing, cleavedcollagen type II and alkaline phosphatase, which are biomarkers ofgrowth, were also produced in the vehicle-treated mice. In addition,there was a one week period of no treatment for the mice in each ofGroups 1 to 4, which possibly diluted out any changes in the cleavedcollagen type II and total alkaline phosphatase levels between theCNP-treated mice in Groups 1-3 and the vehicle-treated mice. There wasno period of no treatment for the CNP-treated mice in Group 5, and thosemice exhibited significantly (p<0.001) greater total alkalinephosphatase level compared to vehicle-treated mice.

Example 15 Dose Responses of CNP Variants in Mice

The effects of varying doses of CNP variants were assessed in wild typeFVB mice.

For the dose study, in two separate studies (S1 and S2), groups of 10mice were administered Pro-Gly-wtCNP37 (“Pro-CNP38”) at 20 and 70nmol/kg subcutaneously once daily for 36 injections. Tail length andbody weight were measured over the course of treatment. Animals weresacrificed at the end of the experiment and bone length assessed.

Tail length of vehicle treated animals was approximately 8 cm at day 36,whereas animals treated with 20 nmol/kg Pro-CNP38 exhibited tail lengthof approximately 8.75 cm and 70 nmol/kg Pro-CNP38 increased tail lengthto approximately 9.5 cm. Administration of Pro-CNP38 in either doseinduces a significant (p<0.05) relative increase in total body lengthcompared to control treated animals, demonstrating approximately a 130%increase in growth velocity at 20 nmol/kg and approximately a 160%increase in growth velocity at 70 nmol/kg Pro-CNP38 (FIG. 69).

Treatment with Pro-CNP 38 also significantly increased bone length inmost of the long bones assessed as well as total naso-anal (body length)of the animal. Table 14 is representative of the relative % increase inbone length in treated animals in comparison to vehicle treated animals.

TABLE 14 Naso- Pro-CNP38 Tail Anal LV4-6 Femur Tibia Humerus Ulna 20nmol/kg  8%*  7%* 10%*  7%* 4%* 3%* 3% 70 nmol/kg 16%* 13%* 13%* 10%*7%* 4%* 5%* Relative % increase, *p < 0.05 ANNOVA (Dunnett's) v vehicle

Bone mineral density (BMD) and bone mineral content (BMC) were alsoevaluated after administration of Pro-CNP38 at different doses. Results(FIG. 70) showed that administration of Pro-CNP38 at 70 nmol/kgsignificantly decreased bone mineral density (FIG. 70A) and increasedbone mineral content (FIG. 70B), suggesting there is a delay in bonemineralization in treated animals, but the mineralization process itselfis not adversely affected by treatment with CNP.

There were no significant changes in organ weight between Pro-CNP38 orvehicle treated animals.

Bioanalytical Studies of Dose Response in Mice

Bioanalytical studies were carried out to measure markers of CNPactivity after in vivo administration of varying doses of Pro-CNP38.Plasma cGMP levels, serum levels of collagen type II and serum levels ofalkaline phosphatase from in vivo samples were analyzed. Wild type micewere administered 20 and 70 nmol/kg Gly-wtCNP37 (“CNP38”), 20 and 70nmol/kg Pro-Gly-wtCNP37 (“Pro-CNP38”) and 70 and 200 nmol/kg ofGHKSEVAHRFK-wtCNP27 (“HSA-CNP27”) (SEQ ID NO: 144) subcutaneously oncedaily for 36 days. Plasma was collected 15 minutes after the lastinjection on day 36 and mice were sacrified 24 hr later. At sacrifice,terminal bleed serum was collected and used for biomarker analysis asdescribed previously.

FIG. 71 shows that 20 nmol/kg CNP38 and 70 nmol/kg HSA-CNP27signficantly increased plasma cGMP (p<0.01), raising plasma cGMP levelsto approximately 300 pmol and 400 pmol, respectively. Adminsitration of70 nmol/kg CNP38 increased cGMP to approximately 500 pmol (p<0.01),while administration of 70 nmol/kg Pro-CNP38 increased cGMP toapproximately 575 pmol (p<0.001). Administration of 200 nmol/kgHSA-CNP27 increased cGMP to approximately 675 pmol (p<0.001).

CNP variants also significantly increased serum levels of cleavedcollagen type II (FIG. 72). CNP38 at 20 nmol/kg increased collagen toapproximately 9 pg/ml (p<0.05), CNP38 at 70 nmol/kg increased collagento approximately 8 pg/ml (p<0.05), Pro-CNP38 at 20 nmol/kg increasedcollagen to approximately 12 pg/ml (p<0.05), Pro-CNP38 at 70 nmol/kgincreased collagen to approximately 16 pg/ml (p<0.05), HSA-CNP27 at 70nmol/kg increased collagen to approximately 10 pg/ml, and HSA-CNP27 at200 nmol/kg increased collagen to approximately 10 pg/ml (p<0.05).

Serum alkaline phosphatase (AP) levels also increased afteradministration of CNP variants (FIG. 73). CNP38 at 20 nmol/kg increasedAP to approximately 130 IU/L, CNP38 at 70 nmol/kg increased AP toapproximately 160 IU/L (p<0.001), Pro-CNP38 at 20 nmol/kg increased APto approximately 155 IU/L (p<0.001), Pro-CNP38 at 70 nmol/kg increasedAP to approximately 180 IU/L (p<0.001), HSA-CNP27 at 70 nmol/kgincreased AP to approximately 120 IU/L, and HSA-CNP27 at 200 nmol/kgincreased AP to approximately 140 IU/L (p<0.01). Table 15 illustratesthe percent of total AP that is bone specific.

TABLE 15 Total AP Bone-specific AP % of Total IU/L IU/L Alk Phos Vehicle109.8 24.7 22.5 CNP38 (20 nmol/kg) 135.9 47.9 35.2 CNP38 (70 nmol/kg)166.3 63.5 38.2 Pro-CNP38 (20 nmol/kg) 159.9 65.9 41.2 Pro-CNP38 (70nmol/kg) 183.6 78.4 42.7 HSA-CNP27 (20 nmol/kg) 121.4 51.7 42.6HSA-CNP27 (70 nmol/kg) 136.1 76.5 56.2

Analysis of anti-CNP antibodies showed that only HSA-CNP27 elicited anIgG antibody response in mice.

The results above illustrate that administration of CNP variantsincreases the concentration of collagen type II and alkaline phosphatasein serum, indicating that CNP increases factors relevant for increasedbone growth, and suggest that administration of CNP variants at doses aslow as 20 nmol/kg are effective in increasing bone growth in vivo.

cGMP Response After Different Dose Regimens

Bioanalytical analysis was also assessed at different times afteradministration of Pro-Gly-wtCNP37 (“Pro-CNP38”) to wild type CD-1 mice,8-10 weeks old, (n=3 per treatment group). Pro-CNP38 was given in asingle subcutaneous dose of 200 nmol/kg and levels of cGMP measured at15 minutes, 3 hours, 1 day, 2 days and 3 days post injection in theplasma, epiphysis, cortical bone (marrow removed), lung and brain. Bloodwas collected on K2EDTA. Tibial and femoral epiphysis and cortical bone,ear pinna, brains, kidneys and lungs were harvested, placed in boilingwater for 5 minutes, then frozen to −70° C. Both plasma and tissue wereassayed for cGMP (Cayman Chemical cyclic GMP ELISA kit).

Results showed that cGMP levels increased at 15 minutes post injectionin plasma (approximately 1300 pmol/ml) and epiphysis (approximately 2.5pmol/ml/mg). By 3 hours, plasma levels had decreased approximately tocontrol levels, while levels in the epiphysis were approximately 3 foldlower than cGMP levels at 15 minutes post injection, but higher thancontrol levels. Levels of cortical bone increased to approximately 0.5pmol/ml/mg at 15 minutes and remained at this level at 3 hours. Levelsof cGMP at all timepoints were back to control levels by 1 day afterinjection. Little to no cGMP was detected in lung or brain at anytimepoint.

cGMP levels were also measured in mice administered multiple injectionsof Pro-CNP38. Groups of mice (n=3) were given Pro-CNP38 as follows: 20nmol/kg single dose, subcutaneously; 200 nmol/kg single dose,subcutaneously; 20 nmol/kg subcutaneously on days 0 and 1; 200 nmol/kgsubcutaneously on days 0 and 1; 20 nmol/kg subcutaneously on days 0 and3; 200 nmol/kg subcutaneously on days 0 and 3. Mice were sacrificed 15minutes after the final dose of Pro-CNP38 and plasma levels of cGMPanalyzed. There does not appear to be a modulation of the plasma cGMPsignal due to the different dose regimens. cGMP responses in thecartilage are also investigated.

Evaluation of Potential Desensitization of NPR-B Receptor

Histological analysis shows that CNP, when administered daily at 200nmol/kg, accumulates in the growth plate of animals, based on increasedCNP immunoreactivity. It is possible that this accumulation, or dailystimulation of the CNP receptor, in the growth plate could desensitizethe CNP receptor.

To determine if multiple dosing desensitizes the NPR-B receptor invitro, normal human articular chondrocytes were cultured withPro-Gly-wtCNP37 (“Pro-CNP38”) for varying times and cGMP secretionmeasured.

Primary normal human chondrocytes isolated from articular cartilage werecultured as recommended by the supplier (Lonza). At 60-80% confluencethe chondrocytes were treated with 1 uM Pro-CNP38 twice, with increasingamounts of time between treatments (first treatment at time 0, then at15 min, 30 min, 60 min, 2 hrs, 3 hrs, 4 hrs, 6 hrs after the initialtreatment). In the following experiment the treatments were eitherapplied twice, with increasing amounts of time between treatment (firsttreatment at time 0, then at 6, 16, 24, 48 hrs after initial treatment)or only once, in parallel to the second treatment (only at 6, 16, 24 and48 hrs-naïve responses). Treatments were either applied for 15 min only(acute treatment), or for the duration of the experiment (chronictreatment when CNP was left in the medium). Cell lysates and conditionedmedium were collected and analyzed for total cGMP secretion (MolecularDevices ELISA).

In a short term experiment, cells were stimulated with Gly-wtCNP37(“CNP38”) 1 uM two times for 15 minutes (acute treatment), or two timeswith CNP38 throughout culture (chronic treatment). The periods betweentreatments were 15 min, 30 min, 60 min, 2 hrs, 3 hrs, 4 hrs, and 6 hrs.Peak cGMP stimulation was obtained by treating cells once only, at thelast time point of the experiment, (6 hours; >0.1 pmol/well in the acuteexperiment and 0.2 pmol/well in the chronic experiment). In the acuteexperiment, when the cells are treated twice, the response decreases to0.1 pmol/well if the cells are treated at time 0 and then again at 15minutes. In the acute experiment, when the cells are treated twice, theresponse decreases to approximately 0.5 pmol/well if the cells aretreated at time 0 and again at 30 min, 60 min, 2, 3, and 4 hours. In thechronic experiment, when the cells are treated twice, the responsedecreases to approximately 0.16 pmol/well if the cells are treated attime 0 and at 15 minutes. In the chronic experiment, when the cells aretreated twice, the response decreases to approximately 0.6 pmol/well ifthe cells are treated at time 0 and again at 30 min, 60 min, or 2 hours.In the chronic experiment, when the cells are treated twice, theresponse decreases to <0.5 pmol/well if the cells are treated at time 0and again at 2, 4 or 6 hours.

Long term studies were also carried out. For acute treatment, 1 uM CNP38was added to cell culture as described above. For chronic treatment, 1uM CNP38 was added to cell culture throughout the experiment duration asdescribed above. Results indicate that the NPR-B receptor candesensitize after repeated, daily CNP administration in vitro, and 48hours between doses is sufficient to recover to 60% of the maximialNPR-B response to CNP38, if the CNP is removed after the first dose, andto <40% if the CNP is incubated throughout the experiment (FIG. 74).

To evaluate whether treatment with a CNP variant desensitizes the NPR-Breceptor in vivo, experiments were conducted with wild-type mice. CD-1male mice 8-10 weeks of age were injected subcutaneously with vehiclecontrol or Pro-Gly-wtCNP37 (“Pro-CNP38”) at 200 nmol/kg on theappropriate days. The mice were either injected with Pro-CNP38 daily forup to 8 days, or were injected with Pro-CNP38 on the first day, thefirst and second days, or the first and third days of the study. Fifteenminutes following the final injection, the mice (n=3 per treatmentgroup) were deeply anesthetized and ex-sanguinated via thoracotomy andaortic cannulation. Circulating blood was flushed from the body with PBSvia an aortic cannula. The kidneys and right tibias, right femurs andleft femurs were collected, boiled in water for 5 minutes and/or snapfrozen in liquid nitrogen and stored on dry ice or in a −70° C. freezeruntil cGMP assay. For estimation of cGMP production in cartilage, distalfemurs and/or proximal tibias were dissected, weighed and pulverizedusing Covaris Cryoprep CP02. Powdered samples were homogenized using aCovaris E210 sonicator in 5% perchloric acid, and neutralized in 60%KOH. Samples were then centrifuged at 10,000 rpm at 4° C. for 5 minutes,and the supernatant was used for the cGMP assay (Cyclic GMP EnzymeImmunoassay Kit, Cayman, Mich.). Additionally, left tibias werecollected, fixed in 10% normal buffered formalin, and saved for furtherimmunohistochemical analysis.

FIG. 75A shows that repeated daily treatment of wild-type mice with 200nmol/kg of Pro-CNP38 for 1, 4, 6, 7 and 8 days did not result indesensitization of the cGMP response. To the contrary, potentiation ofthe cGMP response was observed after 4 days of daily treatment. Furtherdaily treatment resulted in a plateau of the cGMP reponse, up to 8 days.The results indicate that daily treatment of wild-type mice with 200nmol/kg of Pro-CNP38 for up to 8 days do not desensitize the cGMPresponse.

The kinetics of the cGMP response in distal femoral cartilage aftertreatment of the wild-type mice with Pro-CNP38 was also investigated.Treatment of the mice once a day for two days potentiated the cGMPresponse to Pro-CNP38, compared with the cGMP response to a singletreatment (FIG. 75B). When the mice were treated on the first and thirddays, but not on the first and second days, the cGMP response after thesecond treatment (on the third day) was substantially similar to thecGMP response observed after a single treatment on day one (FIG. 75B).The results suggest that dosing on consecutive days is beneficial forpotentiating the cGMP response to Pro-CNP38 in this mouse study.

Activation of NPR-B in Different Tissues

To evaluate potential activation of NPR-B in different tissues by a CNPvariant, wild-type male CD-1 mice were injected with 200 nmol/kg ofGly-CNP37, and the cGMP response was measured in certain tissues atdifferent time points. Two mice were used for each treatment group. Eachmouse received one subcutaneous injection of Gly-CNP37 or vehiclecontrol. Fifteen, 30 or 60 minutes or 3 hours following the injection,the mice were deeply anesthetized and ex-sanguinated via thoracotomy andaortic cannulation. Circulating blood was removed by a PBS flush with anaortic cannula. Heart, liver, lung, kidney, ear pinna, aorta and brainwere collected. All tissues were boiled in water for 5 minutes, finelydissected (including flushing of marrow from femoral cortices with PBS),weighed, cooled in liquid nitrogen, and pulverized in a BioPulverizer.The resulting powdered samples were homogenized with a Polytron in 6%pre-chilled perchloric acid and neutralized with 60% KOH. Samples werethen centrifuged at 10,000 rpm and 4° C. for 5 minutes, and thesupernatant was used for the assay of cGMP (Cyclic GMP EnzymeImmunoassay Kit, Cayman Chemical Company, Ann Arbor, Mich.). The resultswere normalized for tissue weight.

Secretion of cGMP in response to treatment with Gly-CNP37 (“CNP” in FIG.76) was detectable in distal femurs (cartilage and bone), femoralcortices (bone), ear pinna (cartilage), and kidney (FIGS. 76A-D).Maximal cGMP responses in those tissues were observed 15 min aftertreatment. Liver, heart, lung and brain tissues did not exhibitappreciable cGMP secretion in response to Gly-CNP37 relative to vehiclecontrol at the studied time points (FIGS. 76E-H). The results indicatethat treatment with 200 nmol/kg of Gly-CNP37 stimulated cGMP secretionin cartilage, bone and renal tissues.

Example 16 Dose Responses of CNP Variants in Monkeys

The effects of the CNP variant Pro-Gly-CNP37 on bone growth and thelevels of bone growth-related biomarkers are evaluated in cynomolgusmonkeys. Eight normal juvenile cynomolgus monkeys (about 2.5 years ofage at the start of the on-going study) are subcutaneously injecteddaily with 10 or 36 μg/kg/day of Pro-Gly-CNP37 (n=4 per dose group).Four such monkeys are administered vehicle as control. The total lengthof treatment is 6 months. Various measures of growth plate expansion andbone growth are made by digital X-ray and magnetic resonance imaging,and by measurement of limb and body lengths externally. Blood and urinesamples are collected periodically for clinical pathology andmeasurement of levels of Pro-Gly-CNP37 and biomarkers. Followingtermination of the study, gross pathology is performed and tissuesamples are evaluated histologically for assessment of efficacy andsafety.

Data obtained thus far in the on-going study show that both doses ofPro-Gly-CNP37 have increased growth plate width by digital X-ray (FIG.77), increased right and left tibia lengths by digital X-ray (FIGS. 78Aand B), increased leg length by external measurement (FIG. 79),increased arm length by external measurement (FIG. 80), increased bodylength by external measurement (FIG. 81), and increased the serum levelof alkaline phosphatase, a biomarker for bone formation (FIG. 82). Thedata demonstrate that Pro-Gly-CNP37 can stimulate bone growth in normaljuvenile cynomolgus monkeys at hemodynamically acceptable doses.

Example 17 Effects of CNP Variants on Cardiovascular System in Mice

Natriuretic peptides such as CNP have been reported to affect thecardiovascular system. Wang et al. (Eur J Heart Fail. 9:548-57. 2007)describe that CNP has been shown to have a cardioprotective effect inpreventing myocardial ischaemia/reperfusion injury and improving cardiacremodelling after myocardial infarction in rats. Wang demonstrated thatmice overexpressing CNP have reduced incidence of cardiac hypertrophycaused by myocardial infarction. Additionally, CNP has been shown tocause endothelium-independent vasodilation (M. Honing et al.,Hypertension, 37:1179-1183 (2001)) and therefore may transientlydecrease blood pressure in vivo.

To assess the effects of CNP variants on the cardiovascular system, theblood pressure and heart rate in anesthetized wild-type FVB mice isstudied following subcutaneous injection of the variants.

After a pilot study to define a broad dose range of cardiovascularactivity, a dose-response study is conducted to examine the effects ofthree different dose levels of each CNP variant. Three male FVB miceaged 8 weeks comprise each treatment group. Doses are administeredsubcutaneously to anesthetized mice, and systolic, diastolic and meanarterial pressure (MAP), as well as heart rate, are monitored viaimplanted intraarterial pressure transducers.

Example 18 Formulation of CNP Variants

CNP preformulation studies were carried out to assess the stability ofCNP variant Gly-wtCNP37 (“CNP38”) at different pH's (pH 3, 4, 5, 6, 7and 8) and temperatures (5° C., 25° C., and 40° C.) over time. CNP38exhibited greater stability at pH 4-6 than at the other pH's in thestudies. CNP38 was stable at 5° C. at pH 4-6, with ≧about 95% of CNP38remaining after 15 weeks. When the temperature was raised to 25° C., atpH 4 about 85% of CNP38 remained after 15 weeks, at pH 5 about 85%remained after 15 weeks, and at pH 6 about 80% remained after 15 weeks.When the temperature was raised to 40° C., at pH 4 about 55-60% of CNP38remained after 15 weeks, at pH 5 about 65% remained after 15 weeks, andat pH 6 about 40% remained after 15 weeks. FIG. 83 illustrates theobserved plot of pseudo-first order degradation rate constant (K_(obs))vs. pH from pH 3 to 8 and at 5° C., 25° C. and 40° C. The stability datafor CNP38 in the preformulation studies suggests CNP formulations havinga pH in the range from about 4 to about 6. An acidic pH (e.g., pH≦about6) can promote the stability of a CNP variant by, e.g., minimizing oravoiding deamidation of asparagine and/or glutamine residue(s),isomerization of aspartic acid residue(s), or degradation of the CNPvariant by other pathways.

CNP variants can be formulated in pharmaceutical carriers foradministration to subjects affected by, e.g., bone growth conditions. Insome embodiments, liquid formulations of CNP variants are formulatedaccording to any combinations of the ingredients and their amounts orconcentrations in Table 16.

TABLE 16 Ingredient Class Ingredient Concentration Range Activeingredient CNP variant 10 mg/mL ± 9.9 mg/mL Buffering agent Aceticacid/acetate 10 mM ± 5 mM, or pH 5 ± 1 Buffering agent Citricacid/citrate 10 mM ± 5 mM, or pH 5 ± 1 Isotonicity-adjusting NaCl 140 mM± 20 mM agent Isotonicity-adjusting Sucrose 10% ± 5%  agent Preservativem-Cresol 0.4% ± 0.1% or 0.2% Preservative/anti- Benzyl alcohol 1.5% ±0.5% adsorbent Stabilizer Glycerin (glycerol) 5%-100% (neat)¹ StabilizerMethionine 0.01%-0.2%  Stabilizer Ascorbic acid/ 0.1%-1%   ascorbatesalt Stabilizer Thioglycerol 0.1%-1%   Anti-adsorbent Polysorbate 200.001%-0.5%  Polysorbate 80 0.001%-0.5%  Benzyl alcohol 0.5%-1.5%¹Glycerin is used to minimize or prevent water-driven hydrolysis,deamidation, isomerization or cleavage of CNP variants. For lyophilizedformulations, 4-6% or 6-20% mannitol or sucrose can be substituted forNaCl.

In certain embodiments, lyophilized formulations of CNP variants areprepared from formulations formulated according to any combinations ofthe ingredients and their amounts or concentrations in Table 17.

TABLE 17 Ingredient Class Ingredient Concentration Range Activeingredient CNP variant 10 mg/mL ± 9.9 mg/mL Buffering agent Aceticacid/acetate 10 mM ± 5 mM, or pH 5 ± 1 Buffering agent Citricacid/citrate 10 mM ± 5 mM, or pH 5 ± 1 Isotonicity-adjusting Sorbitol 5%± 3% agent/bulking agent Isotonicity-adjusting Mannitol 5% ± 3%agent/bulking agent Isotonicity-adjusting Sucrose 10% ± 5% agent/bulking agent Preservative m-Cresol 0.4% ± 0.2% Preservative/anti-Benzyl alcohol 1.5% ± 0.5% adsorbent Stabilizer Glycerin (glycerol)5%-100% (neat)¹ Stabilizer Methionine 0.01%-0.2%  Stabilizer Ascorbicacid/ 0.1%-1%   ascorbate salt Stabilizer Thioglycerol 0.1%-1%  Anti-adsorbent Polysorbate 20 0.001%-0.5%  Polysorbate 80 0.001%-0.5% Benzyl alcohol 0.5%-1.5% ¹Glycerin is used to minimize or preventwater-driven hydrolysis, deamidation, isomerization or cleavage of CNPvariants.

In certain embodiments, a formulation comprising a CNP variant has a pHof about 3-7, or about 3-6, or about 3.5-6.5, or about 4-6, or about4-5, or about 4.5-5.5. In some embodiments, for pH 4-5.5 a suitablebuffering agent is acetic acid/acetate (e.g., sodium acetate), and forpH 5.5-6 a suitable buffering agent is citric acid/citrate. Citricacid/citrate (e.g., sodium citrate) is also a suitable buffering agentin the range of pH 3-6 or pH 4-6. In certain embodiments, the bufferingagent has a concentration in the formulation of about 2-50 mM, or about2-40 mM, or about 2-30 mM, or about 5-30 mM, or about 2-20 mM, or about5-20 mM, or about 5-15 mM.

To minimize or avoid deamidation of a CNP variant, the variant can beformulated in pharmaceutically acceptable organic cosolvents, such asglycerin, ethanol, and propylene glycol. Because deamidation occurs byhydrolysis, substitution of an organic cosolvent for water minimizescontact of the CNP variant with water. The concentration of one or moreorganic solvents in an organic-aqueous solvent system can be, e.g., fromabout 10% to about 99%, or about 100% if water is not used.

Also to minimize or avoid deamidation of a CNP variant, water can beremoved from the formulation by lyophilization. In some embodiments,lyophilized formulations contain any combinations of the followingcomponents:

-   -   buffer: sodium acetate and acetic acid, or sodium citrate and        citric acid;    -   isotonicity/bulking agent: mannitol (e.g., 3-10%, 2-8% or 4-6%);        -   sucrose (e.g., 6-20%, 5-15% or 8-12%);    -   antioxidants: methionine and/or ascorbic acid with molal ratio        of each antioxidant to CNP variant from about 0.1:1 to about        1:1, or from about 0.5:1 to about 5:1, or from about 1:1 to        about 15:1, or from about 1:1 to about 10:1, or from about 3:1        to about 10:1.

Deamidation can also be minimized or avoided by storing a CNPcomposition (e.g., a liquid formulation or a lyophilized formulation) atlower temperature, such as at about 5° C., 0° C., −10° C., −20° C., −30°C., −40° C., −50° C., −60° C., −70° C., −80° C., −90° C., or −100° C.

To minimize or avoid oxidation of oxidizable residues (e.g., methionine)in a CNP variant, the variant can be formulated with one or moreantioxidants. Exemplary antioxidants include, but are not limited to,methionine, ascorbic acid, and thioglycerol. Oxidation of, e.g.,methionine residues can also be minimized or prevented by purging oxygenfrom a liquid medium (if a liquid formulation) with nitrogen or argon,and/or by purging oxygen from a container or packaging with nitrogen orargon.

In some embodiments, to minimize or prevent adsorption (e.g., adsorptionof a CNP variant to plastic or glass), Polysorbate 20, Polysorbate 80 orbenzyl alcohol, or a combination thereof, is added to a CNP formulation.In certain embodiments, each of the anti-adsorbent(s) is in aconcentration from about 0.001% to about 0.5%, or from about 0.01% toabout 0.5%, or from about 0.1% to about 1%, or from about 0.5% to about1%, or from about 0.5% to about 1.5%, or from about 0.5% to about 2%, orfrom about 1% to about 2%. Exemplary range(s) of anti-adsorbent(s) inthe formulation include without limitation from about 0.001% to about0.5% of Polysorbate 20, from about 0.001% to about 0.5% of Polysorbate80, and/or from about 0.5% to about 1.5% of benzyl alcohol.

In certain embodiments, a liquid CNP formulation comprises, or alyophilized CNP formulation is prepared from a formulation thatcomprises, (1) an acetic acid/acetate (e.g., sodium acetate) bufferhaving a concentration of about 30 mM±5 or 10 mM buffering agent and apH of about 4±0.5 or 1, and (2) benzyl alcohol (e.g., as a preservativeand/or anti-adsorbent) at a concentration of about 1%±0.5%, andoptionally (3) sucrose at a concentration of about 10%±5%.

Example 19 Clinical Evaluation of CNP Variants

The following example provides guidance on the parameters to be used forthe clinical evaluation of compositions comprising CNP22 or variantsthereof in the therapeutic methods of the present disclosure. Asdiscussed herein, CNP22 or variants thereof will be used in thetreatment of disorders responsive to CNP, including disorders of thebone and vascular smooth muscle. Clinical trials will be conducted whichwill provide an assessment of doses of CNP22 or variants thereof forsafety, pharmacokinetics, and initial response of both surrogate anddefined clinical endpoints. The trial will be conducted for a minimum,but not necessarily limited to, 24 weeks to collect sufficient safetyinformation on about 100 evaluable patients. The initial dose for thetrials will vary from about 0.001 to about 1.0 mg/kg/week, or any of thedoses described herein. In the event that the initial dose in this rangedoes not produce a significant direct clinical benefit, the dose shouldbe increased within this range or beyond this range as necessary, andmaintained for an additional minimal period of, but not necessarilylimited to, 24 weeks to establish safety and to evaluate efficacyfurther.

Measurements of safety will include adverse events, allergic reactions,complete clinical chemistry panel (including kidney and liverfunctions), urinalysis, and CBC with differential. In addition, otherparameters relevant to clinical benefit will be monitored. The presentexample also includes the determination of pharmacokinetic parameters ofCNP22 or variants thereof, including absorption, distribution,metabolism, excretion, and half-life and bioavailability in the blood.It is anticipated that such analyses will help relate dose to clinicalresponse.

Methods

Patients will undergo a baseline medical history and physical exam, anda standard set of clinical laboratory tests (including CBC, Panel 20,CH50, and UA). The patients will be followed closely with weekly visitsto the clinic. The patients will return to the clinic for a completeevaluation one week after completing the treatment period. Should doseescalation be required, the patients will follow the same scheduleoutlined above. Safety will be monitored throughout the trial.

Diagnosis and Inclusion Criteria

The patients may be male or female, with a documented diagnosis of apotentially CNP-responsive disorder. A specific example of a potentiallyCNP-responsive, bone-related disorder is achondroplasia, which may beconfirmed by genetic testing and other evidence of an FGFR-3 mutation ordysfunction. The ideal age range of achondroplasia patients will be frominfant (<1 year of age) to pre-adolescent (<13 years of age). A patientwill be excluded from this study if the patient is pregnant orlactating; has received an investigational drug within 30 days prior tostudy enrollment; or has a medical condition, serious intercurrentillness, or other extenuating circumstance that may significantlydecrease study compliance.

Safety

Therapy with CNP22 or variants thereof will be determined to be safe ifno significant acute or chronic drug reactions occur during the courseof the study. The longer-term administration of the drug will bedetermined to be safe if no significant abnormalities are observed inthe clinical examinations, clinical labs, or other appropriate studies.

It has been shown that compared to wild-type CNP22, certain CNP variantsof the disclosure are much more resistant to NEP degradation in vitro,have a much longer plasma half-life and bioavailability in rats,stimulate a much higher level of cGMP production in rats, and/or inducea significantly greater increase in long bone length and body length inachondroplastic mice. Furthermore, it has been shown that short durationdose regimen treatments with CNP22 are nearly as effective as continuousCNP22 treatment in reversing FGF2-induced arrest of chondrocyte growthin vitro. These results, among others described herein, demonstrate theutility of CNP variants of the disclosure in treating CNP-responsiveconditions or disorders such as, e.g., bone-related disorders andvascular smooth muscle disorders.

It is understood that every embodiment of the disclosure describedherein may optionally be combined with any one or more of the otherembodiments described herein. Every patent literature and everynon-patent literature cited herein are incorporated herein by referencein their entirety.

Numerous modifications and variations to the disclosure, as set forth inthe embodiments and illustrative examples described herein, are expectedto occur to those skilled in the art. Consequently only such limitationsas appear in the appended claims should be placed on the disclosure.

What is claimed is:
 1. A variant of C-type natriuretic peptide (CNP)selected from the group consisting of: (SEQ ID NO: 179)GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC (Gly-CNP53);(SEQ ID NO: 185) PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP53); (SEQ ID NO: 190)MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC (Met-CNP53)(SEQ ID NO: 180) DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-53(M48N)].


2. A pharmaceutical composition comprising a CNP variant of claim 1, anda pharmaceutically acceptable excipient, carrier or diluent.
 3. Thecomposition of claim 2, which is a lyophilized formulation prepared froma formulation that comprises a citric acid/citrate buffer or an aceticacid/acetate buffer having a pH from about 4 to about
 6. 4. Thecomposition of claim 3, wherein the lyophilized formulation is preparedfrom a formulation that further comprises (a) an isotonicity-adjustingagent or a bulking agent or (b) an antioxidant.
 5. A method of treatinga bone-related disorder or skeletal dysplasia, comprising administeringa CNP variant to a subject in need thereof, wherein the CNP variant is aCNP variant according to claim 1, and wherein the bone-related disorderor skeletal dysplasia is selected from the group consisting ofachondroplasia, hypochondroplasia, short stature, dwarfism andhomozygous achondroplasia.
 6. A method for recombinant production of aCNP variant, comprising culturing in a medium a host cell comprising afirst polynucleotide encoding a CNP variant polypeptide linked to asecond polynucleotide encoding a cleavable peptide or protein underconditions that result in expression of a fusion polypeptide encoded bythe polynucleotides, wherein the fusion polypeptide comprises the CNPvariant polypeptide directly linked to the cleavable peptide or proteinor indirectly linked thereto via a linker, wherein the CNP variant isselected from the group consisting of: (SEQ ID NO: 179)GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC (Gly-CNP53);(SEQ ID NO: 185) PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP53); (SEQ ID NO: 190)MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC (Met-CNP53);(SEQ ID NO: 180) DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-53(M48N)].


7. The method of claim 6, wherein the cleavable peptide or protein isselected from the group consisting of histidine tags, humantranscription factor TAF12, TAF12 fragments, TAF12 histone fold domain,mutants of TAF12 and fragments thereof, TAF12(C/A), TAF12(D/E),TAF12(4D/4E), TAF12(6D/6E), TAF12(10D/10E), TAF12(C/A & D/E), TAF12(C/A& 4D/4E), TAF12(C/A & 6D/6E), TAF12(C/A & 10D/10E), ketosteroidisomerase, maltose-binding protein, β-galactosidase,glutathione-S-transferase, thioredoxin, chitin-binding domain, BMP-2,BMP-2 mutants, BMP-2(C/A), and mutants and fragments thereof.
 8. Themethod of claim 6, wherein the host cell is bacterial.
 9. The method ofclaim 6, wherein the fusion polypeptide is expressed as a solubleprotein or as an inclusion body.
 10. The method of claim 6, furthercomprising isolating the expressed fusion polypeptide from the host cellor culture medium.
 11. The method of claim 10, further comprisingcontacting the isolated fusion polypeptide with a cleaving agentselected from the group consisting of formic acid, cyanogen bromide(CNBr), hydroxylamine, protein self cleavage, Factor Xa, enterokinase,ProTEV, and SUMO protease.
 12. A CNP variant produced according to themethod of claim 6, wherein the CNP variant is selected from the groupconsisting of: (SEQ ID NO: 179)GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC (Gly-CNP53));(SEQ ID NO: 185) PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP53)); (SEQ ID NO: 190)MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC (Met-CNP53));(SEQ ID NO: 180) DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-53(M48N)]).


13. A method of increasing long bone growth, comprising administering aCNP variant according to claim 1 to a subject in need thereof, where theadministration increases long bone growth.
 14. A CNP variant accordingto claim 1 useful for increasing long bone growth or treatingachondroplasia, hypochondroplasia, short stature, dwarfism, orhomozygous achondroplasia in a subject in need thereof.